Neural basis for fasting activation of the hypothalamic-pituitary-adrenal axis.

Fasting initiates a multitude of adaptations to allow survival. Activation of the hypothalamic-pituitary-adrenal (HPA) axis and subsequent release of glucocorticoid hormones is a key response that mobilizes fuel stores to meet energy demands1-5. Despite the importance of the HPA axis response, the neural mechanisms that drive its activation during energy deficit are unknown. Here, we show that fasting-activated hypothalamic agouti-related peptide (AgRP)-expressing neurons trigger and are essential for fasting-induced HPA axis activation. AgRP neurons do so through projections to the paraventricular hypothalamus (PVH), where, in a mechanism not previously described for AgRP neurons, they presynaptically inhibit the terminals of tonically active GABAergic afferents from the bed nucleus of the stria terminalis (BNST) that otherwise restrain activity of corticotrophin-releasing hormone (CRH)-expressing neurons. This disinhibition of PVHCrh neurons requires γ-aminobutyric acid (GABA)/GABA-B receptor signalling and potently activates the HPA axis. Notably, stimulation of the HPA axis by AgRP neurons is independent of their induction of hunger, showing that these canonical 'hunger neurons' drive many distinctly different adaptations to the fasted state. Together, our findings identify the neural basis for fasting-induced HPA axis activation and uncover a unique means by which AgRP neurons activate downstream neurons: through presynaptic inhibition of GABAergic afferents. Given the potency of this disinhibition of tonically active BNST afferents, other activators of the HPA axis, such as psychological stress, may also work by reducing BNST inhibitory tone onto PVHCrh neurons.

Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at -25 °C for Several Years.

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

Review: Vaspin (SERPINA12) Expression and Function in Endocrine Cells.

Proper functioning of the body depends on hormonal homeostasis. White adipose tissue is now known as an endocrine organ due to the secretion of multiple molecules called adipokines. These proteins exert direct effects on whole body functions, including lipid metabolism, angiogenesis, inflammation, and reproduction, whereas changes in their level are linked with pathological events, such as infertility, diabetes, and increased food intake. Vaspin-visceral adipose tissue-derived serine protease inhibitor, or SERPINA12 according to serpin nomenclature, is an adipokine discovered in 2005 that is connected to the development of insulin resistance, obesity, and inflammation. A significantly higher amount of vaspin was observed in obese patients. The objective of this review was to summarize the latest findings about vaspin expression and action in endocrine tissues, such as the hypothalamus, pituitary gland, adipose tissue, thyroid, ovary, placenta, and testis, as well as discuss the link between vaspin and pathologies connected with hormonal imbalance.

Defects in GnRH Neuron Migration/Development and Hypothalamic-Pituitary Signaling Impact Clinical Variability of Kallmann Syndrome.

Kallmann syndrome (KS) is a combination of isolated hypogonadotropic hypogonadism (IHH) with olfactory dysfunction, representing a heterogeneous disorder with a broad phenotypic spectrum. The genetic background of KS has not yet been fully established. This study was conducted on 46 Polish KS subjects (41 males, 5 females; average age: 29 years old). The studied KS patients were screened for defects in a 38-gene panel with next-generation sequencing (NGS) technology. The analysis revealed 27 pathogenic and likely pathogenic (P/LP) variants, and 21 variants of uncertain significance (VUS). The P/LP variants were detected in 20 patients (43.5%). The prevalence of oligogenic P/LP defects in selected genes among KS patients was 26% (12/46), whereas the co-occurrence of other variants was detected in 43% (20 probands). The examined KS patients showed substantial genotypic and phenotypic variability. A marked difference in non-reproductive phenotypes, involving defects in genes responsible for GnRH neuron development/migration and genes contributing to pituitary development and signaling, was observed. A comprehensive gene panel for IHH testing enabled the detection of clinically relevant variants in the majority of KS patients, which makes targeted NGS an effective molecular tool. The significance of oligogenicity and the high incidence of alterations in selected genes should be further elucidated.

Vitamin K in Vertebrates' Reproduction: Further Puzzling Pieces of Evidence from Teleost Fish Species.

Vitamin K (VK) is a fat-soluble vitamin that vertebrates have to acquire from the diet, since they are not able to de novo synthesize it. VK has been historically known to be required for the control of blood coagulation, and more recently, bone development and homeostasis. Our understanding of the VK metabolism and the VK-related molecular pathways has been also increased, and the two main VK-related pathways-the pregnane X receptor (PXR) transactivation and the co-factor role on the γ-glutamyl carboxylation of the VK dependent proteins-have been thoroughly investigated during the last decades. Although several studies evidenced how VK may have a broader VK biological function than previously thought, including the reproduction, little is known about the specific molecular pathways. In vertebrates, sex differentiation and gametogenesis are tightly regulated processes through a highly complex molecular, cellular and tissue crosstalk. Here, VK metabolism and related pathways, as well as how gametogenesis might be impacted by VK nutritional status, will be reviewed. Critical knowledge gaps and future perspectives on how the different VK-related pathways come into play on vertebrate's reproduction will be identified and proposed. The present review will pave the research progress to warrant a successful reproductive status through VK nutritional interventions as well as towards the establishment of reliable biomarkers for determining proper nutritional VK status in vertebrates.

Mathematical modeling approaches of cellular endocrinology within the hypothalamo-pituitary-gonadal axis.

The reproductive neuroendocrine axis, or hypothalamo-pituitary-gonadal (HPG) axis, is a paragon of complex biological system involving numerous cell types, spread over several anatomical levels communicating through entangled endocrine feedback loops. The HPG axis exhibits remarkable dynamic behaviors on multiple time and space scales, which are an inexhaustible source of studies for mathematical and computational biology. In this review, we will describe a variety of modeling approaches of the HPG axis from a cellular endocrinology viewpoint. We will in particular investigate the questions raised by some of the most striking features of the HPG axis: (i) the pulsatile secretion of hypothalamic and pituitary hormones, and its counterpart, the cell signaling induced by frequency-encoded hormonal signals, and (ii) the dual, gametogenic and glandular function of the gonads, which relies on the tight control of the somatic cell populations ensuring the proper maturation and timely release of the germ cells.

Functional Pituitary Networks in Vertebrates.

The pituitary is a master endocrine gland that developed early in vertebrate evolution and therefore exists in all modern vertebrate classes. The last decade has transformed our view of this key organ. Traditionally, the pituitary has been viewed as a randomly organized collection of cells that respond to hypothalamic stimuli by secreting their content. However, recent studies have established that pituitary cells are organized in tightly wired large-scale networks that communicate with each other in both homo and heterotypic manners, allowing the gland to quickly adapt to changing physiological demands. These networks functionally decode and integrate the hypothalamic and systemic stimuli and serve to optimize the pituitary output into the generation of physiologically meaningful hormone pulses. The development of 3D imaging methods and transgenic models have allowed us to expand the research of functional pituitary networks into several vertebrate classes. Here we review the establishment of pituitary cell networks throughout vertebrate evolution and highlight the main perspectives and future directions needed to decipher the way by which pituitary networks serve to generate hormone pulses in vertebrates.

Effect of oestrogen-dependent vasopressin on HPA axis in the median eminence of female rats.

The median eminence (ME) anatomically consists of external (eME) and internal (iME) layers. The hypothalamic neurosecretory cells terminate their axons in the eME and secrete their neurohormones regulating anterior pituitary hormone secretion involved in stress responses into the portal vein located in the eME. Magnocellular neurosecretory cells (MNCs) which produce arginine vasopressin (AVP) and oxytocin in the paraventricular (PVN) and supraoptic nuclei (SON) terminate their axons in the posterior pituitary gland (PP) through the iME. Here, we provide the first evidence that oestrogen modulates the dynamic changes in AVP levels in the eME axon terminals in female rats, using AVP-eGFP and AVP-DREADDs transgenic rats. Strong AVP-eGFP fluorescence in the eME was observed at all oestrus cycle stages in adult female rats but not in male transgenic rats. AVP-eGFP fluorescence in the eME was depleted after bilateral ovariectomy but re-appeared with high-dose 17ß-oestradiol. AVP-eGFP fluorescence in the MNCs and PP did not change significantly in most treatments. Peripheral clozapine-N-oxide administration induced AVP-DREADDs neurone activation, causing a significant increase in plasma corticosterone levels in the transgenic rats. These results suggest that stress-induced activation of the hypothalamic-pituitary-adrenal axis may be caused by oestrogen-dependent upregulation of AVP in the eME of female rats.

Somatostatin receptor subtype 5 modifies hypothalamic-pituitary-adrenal axis stress function.

Pituitary corticotroph somatostatin receptor subtype 5 (SSTR5) signals to inhibit adrenocorticotrophin (ACTH) secretion. As ACTH deficiency results in attenuated adrenal cortisol production and an impaired stress response, we sought to clarify the role of SSTR5 in modifying the hypothalamic/pituitary/adrenal (HPA) axis. We generated Tg HP5 mice overexpressing SSTR5 in pituitary corticotrophs that produce the ACTH precursor proopiomelanocortin (POMC). Basal ACTH and corticosterone were similar in HP5 and WT mice, while HP5 mice showed attenuated ACTH and corticosterone responses to corticotrophin releasing hormone (CRH). HP5 mice exhibited attenuated corticosterone responses upon a restraint stress test and inflammatory stress following LPS injection, as well as increased anxiety-like and depressive-like behavior on open field and forced swim tests. Pituitary corticotroph CRH receptor subtype 1 (CRHR1) mRNA expression and ACTH responses to CRH were also attenuated in HP5 mice. In AtT20 cells stably overexpressing SSTR5, CRHR1 expression and cAMP response to CRH were reduced, whereas both were increased after SSTR5 KO. In elucidating mechanisms for these observations, we show that SSTR5-induced miR-449c suppresses both CRHR1 expression and function. We conclude that corticotroph SSTR5 attenuates HPA axis responses via CRHR1 downregulation, suggesting a role for SSTR5 in the pathogenesis of secondary adrenal insufficiency.

Interaction of Melatonin and BMP-6 in Ovarian Steroidogenesis.

The bone morphogenetic protein (BMP) system in the ovary plays a physiological role as a luteinization inhibitor in growing follicles. BMP-6 secreted from oocytes and granulosa cells can exert an inhibitory effect on follicle-stimulating hormone (FSH) actions by suppressing adenylate cyclase activity downstream of the FSH receptor. The inhibition of FSH-induced progesterone production by BMP-6 is impaired by melatonin treatment in granulosa cells. Intracellular Smad signaling induced by BMP-6 is suppressed by melatonin, suggesting that melatonin has a regulatory role in BMP receptor signaling in granulosa cells. Since the expression of BMP-6 in granulosa cells is increased in patients with polycystic ovary syndrome, melatonin may play an important role in the maintenance of progesterone production by suppressing BMP-6 signaling, leading to the preservation of ovarian function.

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study.

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic-pituitary-gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1-expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP-expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons-that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates.

Cells have sex chromosomes and circadian clocks: Implications for organismal level functions.

A great number of stakeholders have a keen interest in issues surrounding sex differences. These participants in the discourse often use the same evidence to draw opposite conclusions, with implications for individuals and society as a whole. One part of the maelstrom and associated emotionality derives from confounds between the concepts of "sex" vs. "gender", even among professionals. Here, the oft-repeated point is made that evidence for gender differences can't be derived from the animal research, once the generally accepted conception of gender as a process unique to humans, is acknowledged. Nevertheless, considered at a more general level, the developmental and epigenetic mechanisms that give rise to differences in behavior among individuals and groups is exquisitely explored in animal studies but relatively poorly in research on humans. The focus on animal research here, starts with the fact that virtually each cell of the body has sex chromosomes (XX and XY), along with the intracellular genetic and cytoplasmic mechanisms associated with circadian (circa-about, dies-day) timing. The consequences of these sex×circadian interactions for physiology and behavior at cellular and higher levels of organization are considered in systems where compelling evidence is available. These include sex differences in the circadian timing system, the hypothalamic-pituitary-adrenal (HPA) axis, and in metabolism. The evidence highlights sex differences in cells throughout the body and thus has implications for higher level processes and systems such as sleep/wake patterns. In a more general sense, they point to mechanisms that could give rise to gender differences. In summary, the viewpoint presented here is that the circadian timing system can be used very elegantly to explore the contributions of genetic and hormonal sex differences on biological systems at many levels.

Alteration in the relationship between tanycytes and gonadotrophin-releasing hormone neurosecretory terminals following long-term metabolic manipulation in the sheep.

The activity of the hypothalamic-pituitary gonadal axis is influenced by energy reserves, such that an increase or a decrease in adiposity may perturb the secretion and action of gonadotrophin-releasing hormone (GnRH). This is considered to be a result of the signalling of hormones such as leptin, which act upon neuronal systems controlling GnRH secretion. Other work shows plasticity in the relationship between tanycytes and GnRH neurosecretory terminals in the median eminence across the oestrous cycle and we hypothesised that a similar plasticity may occur with altered metabolic status. We studied Lean, Normal and Fat ovariectomised ewes, which displayed differences in gonadotrophin status, and investigated the relationship between tanycytes and GnRH neuroterminals. Under both Lean and Fat conditions, an altered anatomical arrangement between these two elements was observed in the vicinity of the blood vessels of the primary plexus of the hypophysial portal blood system. These data suggest that such plasticity is an important determinant of the rate of secretion of GnRH in animals of differing metabolic status and that this also contributes to the relative hypogonadotrophic condition prevailing with metabolic extremes.

Developmental Origins of Hypothalamic Cells Controlling Reproduction.

The hypothalamus-pituitary-gonadal (HPG) axis is the most critical modulator of reproductive function. Genetic or environmental insults to the HPG axis during developmental windows can persist into adulthood, and processes such as gonadal hormone synthesis, timing of puberty, and fertility can be affected. At the level of the hypothalamus, multiple regions develop at different times and are under the control of a concert of signaling pathways and transcription factors required for their patterning and maturation. In this review, we highlight factors and pathways involved in specification and ultimate differentiation of neuronal and other cellular subtypes of the hypothalamus contributing to the HPG axis. Specifically, we discuss development of the arcuate and anteroventral periventricular nuclei, as well as forebrain development as it relates to reproductive function. Precise control of kisspeptin and gonadotropin-releasing hormone neuron, as well as tanycyte, development is necessary for understanding and ultimately treating developmental disruptions resulting in infertility.

Identification of an endocannabinoid system in the rat pars tuberalis-a possible interface in the hypothalamic-pituitary-adrenal system?

Endocannabinoids (ECs) are ubiquitous endogenous lipid derivatives and play an important role in intercellular communication either in an autocrine/paracrine or in an endocrine fashion. Recently, an intrinsic EC system has been discovered in the hypophysial pars tuberalis (PT) of hamsters and humans. In hamsters, this EC system is under photoperiodic control and appears to influence the secretion of hormones such as prolactin from the adenohypophysis. We investigate the EC system in the PT of the rat, a frequently used species in endocrine research. By means of immunocytochemistry, enzymes involved in EC biosynthesis, e.g., N-arachidonoyl-phosphatidylethanolamine-phospholipase D (NAPE-PLD) and diacylglycerol lipase α (DAGLα) and enzymes involved in EC degradation, e.g., fatty acid amide hydrolase (FAAH) and cyclooxygenase-2 (COX-2), were demonstrated in PT cells of the rat. Immunoreactions (IR) for FAAH and for the cannabinoid receptor CB1 were observed in corticotrope cells of the rat adenohypophysis; these cells were identified by antibodies against proopiomelanocortin (POMC) or adrenocorticotrophic hormone (ACTH). In the outer zone of the median eminence, numerous nerve fibers and terminals displayed CB1 IR. The majority of these were also immunolabeled by an antibody against corticotropin-releasing factor (CRF). These results suggest that the EC system at the hypothalamo-hypophysial interface affects both the CRF-containing nerve fibers and the corticotrope cells in the adenohypophysis. Our data give rise to the hypothesis that, in addition to its well-known role in the reproductive axis, the PT might influence adrenal functions and, thus, the stress response and immune system.

A short-term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA-axis signaling in mice.

There is still uncertainty whether extremely low frequency electromagnetic fields (ELF-EMF) can induce health effects like immunomodulation. Despite evidence obtained in vitro, an unambiguous association has not yet been established in vivo. Here, mice were exposed to ELF-EMF for 1, 4, and 24 h/day in a short-term (1 week) and long-term (15 weeks) set-up to investigate whole body effects on the level of stress regulation and immune response. ELF-EMF signal contained multiple frequencies (20-5000 Hz) and a magnetic flux density of 10 µT. After exposure, blood was analyzed for leukocyte numbers (short-term and long-term) and adrenocorticotropic hormone concentration (short-term only). Furthermore, in the short-term experiment, stress-related parameters, corticotropin-releasing hormone, proopiomelanocortin (POMC) and CYP11A1 gene-expression, respectively, were determined in the hypothalamic paraventricular nucleus, pituitary, and adrenal glands. In the short-term but not long-term experiment, leukocyte counts were significantly higher in the 24 h-exposed group compared with controls, mainly represented by increased neutrophils and CD4 ± lymphocytes. POMC expression and plasma adrenocorticotropic hormone were significantly lower compared with unexposed control mice. In conclusion, short-term ELF-EMF exposure may affect hypothalamic-pituitary-adrenal axis activation in mice. Changes in stress hormone release may explain changes in circulating leukocyte numbers and composition. Bioelectromagnetics. 37:433-443, 2016. © 2016 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Bioactive forms of vitamin D selectively stimulate the skin analog of the hypothalamus-pituitary-adrenal axis in human epidermal keratinocytes.

Ultraviolet radiation B stimulates both the production of vitamin D3 in the skin and the activation of the skin analog of the hypothalamic-pituitary-adrenal axis (HPA) as well as the central HPA. Since the role of vitamin D3 in the regulation of the HPA is largely unknown, we investigated the impact of 1,25(OH)2D3 and its noncalcemic analogs, 20(OH)D3 and 21(OH)pD, on the expression of the local HPA in human epidermal keratinocytes. The noncalcemic analogs showed similar efficacy to 1,25(OH)2D3 in stimulating the expression of neuropeptides, CRF, urocortins and POMC, and their receptors, CRFR1, CRFR2, MC1R, MC2R, MC3R and MC4R. Interestingly, unlike other secosteroids, the activity of 21(OH)pD did not correlate with induction of differentiation, suggesting a separate but overlapping mechanism of action. Thus, biologically active forms of vitamin D can regulate different elements of the local equivalent of the HPA with implications for the systemic HPA.

Neuroanatomical evidence for the involvement of ß-endorphin during reproductive stress response in the fish Oreochromis mossambicus.

Although neuroendocrine regulation of stress response involving hypothalamo-pituitary-adrenal axis is well established in mammals, the mechanism of functioning of endocrine-stress axis is not completely elucidated in fish. Our previous studies suggested a possible role for the opioidergic mediation of reproductive stress response in fish. In the present investigation, by immunocytochemical approach, we studied the distribution of ß-endorphin (ß-EP) secreting neurons in the brain of the tilapia Oreochromis mossambicus exposed to aquacultural stressors. Intensely stained ß-EP immunoreactive neurons were encountered in the nucleus lateralis tuberis (NLT) region during previtellogenic and vitellogenic phases in both controls and in fish exposed to aquacultural stressors. Furthermore, at the end of the prespawning phase in controls, weak staining in ß-EP neurons was accompanied by intensely stained luteinizing hormone (LH) immunoreactive cells in the proximal pars distalis (PPD) of the pituitary gland and a significantly higher gonadosomatic and hepatosomatic indices, suggesting the attenuation of inhibitory effect of ß-EP on reproductive axis prior to spawning. However, in fish exposed to stressors, several darkly stained ß-EP immunoreactive cells with dense fibre projections towards the hypothalamo-hypophysial tract were concomitant with weakly immunoreactive LH content in the PPD of the pituitary gland and a significantly lower gonadosomatic and hepatosomatic indices compared to those of controls. These results suggest that stress-induced activation of ß-EP secreting neurons in the NLT region might lead to the inhibition of LH secreting cells-ovary axis in fish.

Immediate and lasting effects of chronic daily methamphetamine exposure on activation of cells in hypothalamic-pituitary-adrenal axis-associated brain regions.

RATIONALE: Chronic methamphetamine (MA) abuse leads to dependence and symptoms of withdrawal after use has ceased. Negative mood states associated with withdrawal, as well as drug reinstatement, have been linked to drug-induced disruption of the hypothalamic-pituitary-adrenal (HPA) axis. However, effects of chronic MA exposure or acute MA exposure following withdrawal on neural activation patterns within brain regions that regulate the HPA axis are unknown. OBJECTIVES: In this study, neural activation patterns were assessed by quantification of c-Fos protein in mice exposed to different regimens of MA administration. METHODS: (Experiment 1) Adult male mice were treated with MA (5 mg/kg) or saline once or once daily for 10 days. (Experiment 2) Mice were treated with MA or saline once daily for 10 days and following a 10-day withdrawal period were re-administered a final dose of MA or saline. c-Fos was quantified in brains after the final injection. RESULTS: (Experiment 1) Compared to exposure to a single dose of MA (5 mg/kg), chronic MA exposure decreased the number of c-Fos expressing cells in the paraventricular hypothalamus, dorsomedial hypothalamus, central amygdala, basolateral amygdala, bed nucleus of the stria terminalis (BNST), and CA3 hippocampal region. (Experiment 2) Compared to mice receiving their first dose of MA, mice chronically treated with MA, withdrawn, and re-administered MA, showed decreased c-Fos expressing cells within the central and basolateral amygdala, BNST, and CA3. CONCLUSIONS: HPA axis-associated amygdala, extended amygdala, and hippocampal regions endure lasting effects following chronic MA exposure and therefore may be linked to stress-related withdrawal symptoms.

Osmoregulation requires brain expression of the renal Na-K-2Cl cotransporter NKCC2.

The Na-K-2Cl cotransporter 2 (NKCC2) was thought to be kidney specific. Here we show expression in the brain hypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution-rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABA-mediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats.