The hypothalamo-neurohypophysial system (HNS) is a brain peptidergic neurosecretory apparatus which is composed of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurones and their neuronal processes in the posterior pituitary (PP). In response to specific stimuli, AVP and OXT are secreted into the systemic circulation at the neurovascular interface of the PP, where they act as hormones, but they can also behave as neurotransmitters when released at the somatodendritic compartment or by axon collaterals to other brain regions. Because these peptides are crucial for several physiological processes, including fluid homoeostasis and reproduction, it is of great importance to map the HNS connectome in its entirety in order to understand its functions. In recent years, advances in imaging technologies have provided considerable new information about the HNS. These approaches include the use of reporter proteins under the control of specific promoters, viral tracers, brain-clearing methods, genetically encoded indicators, sniffer cells, mass spectrometry imaging, and spatially resolved transcriptomics. In this review, we illustrate how these latest approaches have enhanced our understanding of the structure and function of the HNS and how they might contribute further in the coming years.

The synthesis of arginine vasopressin (AVP) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus is sensitive to increased plasma osmolality and a decreased blood volume, and thus is robustly increased by both dehydration (increased plasma osmolality and decreased blood volume) and salt loading (increased plasma osmolality). Both stimuli result in functional remodelling of the SON and PVN, a process referred to as functional-related plasticity. Such plastic changes in the brain have recently been associated with altered patterns of DNA methylation at CpG (cytosine-phosphate-guanine) residues, a process considered to be important for the regulation of gene transcription. In this regard, the proximal Avp promoter contains a number of CpG sites and is recognised as one of four CpG islands for the Avp gene, suggesting that methylation may be regulating Avp transcription. In the present study, we show that, in an immortalised hypothalamic cell line 4B, the proximal Avp promoter is highly methylated, and treatment of these cells with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine to demethylate DNA dramatically increases basal and stimulated Avp biosynthesis. We report no changes in the expression of DNA methyltransferases, Dnmt1 and Dnmt3a, whereas there is decreased expression of the demethylating enzyme ten-eleven-translocation 2, Tet2, in the SON by dehydration and salt loading. We found higher methylation of the SON Avp promoter in dehydrated but not salt-loaded rats. By analysis of individual CpG sites, we observed hypomethylation, hypermethylation and no change in methylation of specific CpGs in the SON Avp promoter of the dehydrated rat. Using reporter gene assays, we show that mutation of individual CpGs can result in altered Avp promoter activity. We propose that methylation of the SON Avp promoter is necessary to co-ordinate the duel inputs of increased plasma osmolality and decreased blood volume on Avp transcription in the chronically dehydrated rat.

The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

New Findings What is the central question of this study?The chemokine fractalkine (FKN) and its cognate receptor, CX3CR1, are expressed in the brain, but their distribution and function within the paraventricular nucleus of the hypothalamus (PVN) have not been previously investigated. What is the main finding and its importance?We show that both FKN and CX3CR1 receptor mRNA and protein are expressed in the PVN and that FKN elicits hypotension and tachycardia when exogeneously applied by microinjection, an effect that is attenuated in rats with heart failure. These findings are important because they demonstrate that FKN and its cognate receptor are expressed and play a role in controlling cardiovascular function within the PVN. The paraventricular nucleus (PVN) of the hypothalamus plays an important role in the regulation of sympathetic nerve activity, which is significantly elevated in chronic heart failure (CHF). Fractalkine (FKN) and its cognate receptor, CX3CR1, are constitutively expressed in the central nervous system, but their role and physiological significance are not well known. The aims of the present study were to determine whether FKN plays a cardiovascular role within the PVN and to investigate how the actions of FKN might be altered in CHF. We show that both FKN and CX3CR1 are expressed on neurons in the PVN of rats, suggesting that they may have a physiological function in this brain nucleus. Unilateral microinjection of FKN directly into the PVN of anaesthetized rats elicited a significant dose‐related decrease in blood pressure (1.0 nmol, −5 ± 3 mmHg; 2.5 nmol, −13 ± 2 mmHg; 5.0 nmol, −22 ± 3 mmHg; and 7.5 nmol, −32 ± 3 mmHg) and a concomitant increase in heart rate (1.0 nmol, 6 ± 3 beats min−1; 2.5 nmol, 11 ± 3 beats min−1; 5 nmol, 18 ± 4 beats min−1; and 7.5 nmol, 27 ± 5 beats min−1) compared with control saline microinjections. In order to determine whether FKN signalling is altered in rats with CHF, we first performed quantitative RT‐PCR and Western blot analysis and followed these experiments with functional studies in rats with CHF and sham‐operated control rats. We found a significant increase in CX3CR1 mRNA and protein expression, as determined by quantitative RT‐PCR and Western blot analysis, respectively, in the PVN of rats with CHF compared with sham‐operated control rats. We also found that the blood pressure effects of FKN (2.5 nmol in 50 nl) were significantly attenuated in rats with CHF (change in mean arterial pressure, −6 ± 3 mmHg) compared with sham‐operated control rats (change in mean arterial pressure, −16 ± 6 mmHg). These data suggest that FKN and its receptor, CX3CR1, modulate cardiovascular function at the level of the PVN and that the actions of FKN within this nucleus are altered in heart failure.

Prolonged exposure to environmental stress activates the hypothalamic-pituitary-adrenal (HPA) axis and generally disrupts the hypothalamic-pituitary-gonadal axis. Because CRF expression in the central nucleus of the amygdala (CeA) is a key modulator in adaptation to chronic stress, and central administration of CRF inhibits the hypothalamic GnRH pulse generator, we tested the hypothesis that overexpression of CRF in the CeA of female rats alters anxiety behavior, dysregulates the HPA axis response to stress, changes pubertal timing, and disrupts reproduction. We used a lentiviral vector to increase CRF expression site specifically in the CeA of preweaning (postnatal day 12) female rats. Overexpression of CRF in the CeA increased anxiety-like behavior in peripubertal rats shown by a reduction in time spent in the open arms of the elevated plus maze and a decrease in social interaction. Paradoxically, puberty onset was advanced but followed by irregular estrous cyclicity and an absence of spontaneous preovulatory LH surges associated with proestrous vaginal cytology in rats overexpressing CRF. Despite the absence of change in basal corticosterone secretion or induced by stress (lipopolysaccharide or restraint), overexpression of CRF in the CeA significantly decreased lipopolysaccharide, but not restraint, stress-induced suppression of pulsatile LH secretion in postpubertal ovariectomized rats, indicating a differential stress responsivity of the GnRH pulse generator to immunological stress and a potential adaptation of the HPA axis to chronic activation of amygdaloid CRF. These data suggest that the expression profile of this key limbic brain CRF system might contribute to the complex neural mechanisms underlying the increasing incidence of early onset of puberty on the one hand and infertility on the other attributed to chronic stress in modern human society.

The supraoptic nucleus (SON) is part of the central osmotic circuitry that synthesises the hormone vasopressin (Avp) and transports it to terminals in the posterior lobe of the pituitary. Following osmotic stress such as dehydration, this tissue undergoes morphological, electrical and transcriptional changes to facilitate the appropriate regulation and release of Avp into the circulation where it conserves water at the level of the kidney. Here, the organisation of the whole transcriptome following dehydration is modelled to fit Zipf's law, a natural power law that holds true for all natural languages, that states if the frequency of word usage is plotted against its rank, then the log linear regression of this is -1. We have applied this model to our previously published euhydrated and dehydrated SON data to observe this trend and how it changes following dehydration. In accordance with other studies, our whole transcriptome data fit well with this model in the euhydrated SON microarrays, but interestingly, fit better in the dehydrated arrays. This trend was observed in a subset of differentially regulated genes and also following network reconstruction using a third-party database that mines public data. We make use of language as a metaphor that helps us philosophise about the role of the whole transcriptome in providing a suitable environment for the delivery of Avp following a survival threat like dehydration.

We have previously shown that an acute osmotic stimulation induces the expression of a c‐fos and monomeric red fluorescent protein 1 (mRFP1) fusion transgene in osmosensitive rat brain areas, including the supraoptic (SON) and paraventricular nuclei (PVN). However, the effects of chronic stimuli, such as dehydration, have not been investigated. In the present study, the expression patterns of the c‐fos‐mRFP1 fusion gene in the forebrain and the brainstem of male and female transgenic rats were studied in seven experimental groups: ad lib. water (euhydration), water deprivation for 12, 24 or 48 h (dehydration) and water deprivation for 46 h + ad lib. water for 2, 6 or 12 h (rehydration). The number of cells that express nuclear mRFP1 fluorescence was quantified in the hypothalamus, the circumventricular organs and the brainstem. Compared to the euhydrated state, the number of transgene expressing cells significantly increased in all forebrain areas and in the rostral ventrolateral medulla after dehydration and 2 h of rehydration. In the nucleus of the solitary tract and area postrema, the number of mRFP1 fluorescent cells was markedly increased after 2 h of rehydration. Although the number of mRFP1 fluorescent cells in the organum vasculosum laminae terminalis, median preoptic nucleus and subfornical organ remained significantly increased after 6 h of rehydration, reaching control levels after 12 h of rehydration, the number of mRFP1 fluorescent cells in the SON and the PVN reached control levels after 6 h of rehydration. There were no significant differences between male and female rats. These results show that the expression of the c‐fos‐mRFP1 fusion gene changes in the forebrain and the brainstem not only after acute osmotic stimulation, but also after chronic osmotic stimulation. Interestingly, these studies reveal the differential activation of different neuronal groups over the time course of dehydration and rehydration.

The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady‐state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by transcription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down‐regulation of 11 and up‐regulation of 31 transcription factor transcripts, including members of the activator protein‐1 gene family, namely c‐fos, c‐jun, fosl1 and junD. Because JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and the quantitative reverse transcriptase‐polymerase chain reaction to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24 and 72 h of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double‐staining fluorescence immunohistochemistry with a neurone‐specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin‐containing neurones with markedly less staining seen in oxytocin‐containing MCNs. Furthermore, JunD is highly co‐expressed with c‐Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members.

The nucleus of the solitary tract (NTS), an integral vasomotor region located in the hindbrain, is important for cardiovascular homeostasis. Fractalkine (FKN) and its cognate receptor, CX3CR1, are constitutively expressed in the normal rat brain. The physiological significance of this cytokine and its receptor are not well established. In this study, we sought to identify the expression of FKN and CX3CR1 in subnuclei of the NTS and to elucidate their functional relevance. Using immunohistochemistry, we found expression of FKN and CX3CR1 throughout the entire rostro-caudal axis of the NTS in normal adult male Sprague-Dawley rats. When FKN was unilaterally microinjected directly into the commissural and sub-postremal, but not rostral, NTS, blood pressure and heart rate were significantly decreased when compared with saline controls. The FKN-induced depressor and bradycardic responses were inhibited by pretreatment with a phosphoinositide 3-kinase inhibitor, LY294002. These data suggest that the cytokine, FKN, and its receptor, CX3CR1, may modulate cardiovascular responses in the NTS of normal healthy rats via the phosphoinositide 3-kinase intracellular signaling pathway.

Angiogenesis and vascular regression are critical for the female ovulatory cycle. They enable progression and regression of follicular development, and corpora lutea formation and regression. Angiogenesis in the ovary occurs under the control of the vascular endothelial growth factor-A (VEGFA) family of proteins, which are generated as both pro-(VEGF(165)) and anti(VEGF(165)b)-angiogenic isoforms by alternative splicing. To determine the role of the VEGF(165)b isoforms in the ovulatory cycle, we measured VEGF(165)b expression in marmoset ovaries by immunohistochemistry and ELISA, and used transgenic mice over-expressing VEGF(165)b in the ovary. VEGF(165)b was expressed in the marmoset ovaries in granulosa cells and theca, and the balance of VEGF(165)b:VEGF(165) was regulated during luteogenesis. Mice over-expressing VEGF(165)b in the ovary were less fertile than wild-type littermates, had reduced secondary and tertiary follicles after mating, increased atretic follicles, fewer corpora lutea and generated fewer embryos in the oviduct after mating, and these were more likely not to retain the corona radiata. These results indicate that the balance of VEGFA isoforms controls follicle progression and luteogenesis, and that control of isoform expression may regulate fertility in mammals, including in primates.

The elucidation of the genomes of a large number of mammalian species has produced a huge amount of data on which to base physiological studies. These endeavours have also produced surprises, not least of which has been the revelation that the number of protein coding genes needed to make a mammal is only 22 333 (give or take). However, this small number belies an unanticipated complexity that has only recently been revealed as a result of genomic studies. This complexity is evident at a number of levels: (i) cis-regulatory sequences; (ii) noncoding and antisense mRNAs, most of which have no known function; (iii) alternative splicing that results in the generation of multiple, subtly different mature mRNAs from the precursor transcript encoded by a single gene; and (iv) post-translational processing and modification. In this review, we examine the steps being taken to decipher genome complexity in the context of gene expression, regulation and function in the hypothalamic-neurohypophyseal system (HNS). Five unique stories explain: (i) the use of transcriptomics to identify genes involved in the response to physiological (dehydration) and pathological (hypertension) cues; (ii) the use of mass spectrometry for single-cell level identification of biological active peptides in the HNS, and to measure in vitro release; (iii) the use of transgenic lines that express fusion transgenes enabling (by cross-breeding) the generation of double transgenic lines that can be used to study vasopressin (AVP) and oxytocin (OXT) neurones in the HNS, as well as their neuroanatomy, electrophysiology and activation upon exposure to any given stimulus; (iv) the use of viral vectors to demonstrate that somato-dendritically released AVP plays an important role in cardiovascular homeostasis by binding to V1a receptors on local somata and dendrites; and (v) the use of virally-mediated optogenetics to dissect the role of OXT and AVP in the modulation of a wide variety of behaviours.

We have addressed the question of how different rodent species cope with the life-threatening homeostatic challenge of dehydration at the level of transcriptome modulation in the supraoptic nucleus (SON), a specialised hypothalamic neurosecretory apparatus responsible for the production of the antidiuretic peptide hormone arginine vasopressin (AVP). AVP maintains water balance by promoting water conservation at the level of the kidney. Dehydration evokes a massive increase in the regulated release of AVP from SON axon terminals located in the posterior pituitary, and this is accompanied by a plethora of changes in the morphology, electrophysiological properties, biosynthetic and secretory activity of this structure. Microarray analysis was used to generate a definitive catalogue of the genes expressed in the mouse SON, and to describe how the gene expression profile changes in response to dehydration. Comparison of the genes differentially expressed in the mouse SON as a consequence of dehydration with those of the rat has revealed many similarities, pointing to common processes underlying the function-related plasticity in this nucleus. In addition, we have identified many genes that are differentially expressed in a species-specific manner. However, in many cases, we have found that the hyperosmotic cue can induce species-specific alterations in the expression of different genes in the same pathway. The same functional end can be served by different means, via differential modulation, in different species, of different molecules in the same pathway. We suggest that pathways, rather than specific genes, should be the focus of integrative physiological studies based on transcriptome data.

Affymetrix microarray studies have suggested that the expression of the gene encoding the orphan G protein-coupled receptor GPR182 is up-regulated in the rostral ventrolateral medulla (RVLM) of adult spontaneously hypertensive (SHR) rats in comparison with matched normotensive Wistar control rats. We thus hypothesized that RVLM GPR182 may have a role in the aetiology of high blood pressure in the SHR. To test this hypothesis, we suppressed the expression of GPR182 in the RVLM using Adenoviral vectors expressing specific short hairpin RNAs (shRNAs).

Adrenomedullin (ADM) increases mean arterial pressure and sympathetic nerve activity (SNA) when microinjected into the rostral ventrolateral medulla (RVLM) of anesthetized normotensive rats (Xu and Krukoff, 2007; AJP, 287, p R729). In conditions of hypoxia, ADM expression is elevated in the brain (Serrano et al., 2008; Brain Pathology, 18, p 434). Since acute intermittent hypoxia (10% O2) induces long term facilitation (LTF) of SNA in anesthetized rats (Xing & Pilowski, 2010; J. Physiol., 588, p 3075), we hypothesized that the combination of ADM injected into the RVLM with multiple peripheral chemoreflex activation induces LTF of SNA of rats submitted to chronic intermittent hypoxia (CIH). For 10 days male Wistar rats (19–21 days) were submitted to CIH (6% O2 for 40 s, every 9 min, 8 h/day, n=7) or maintained in normoxia (controls, n=8). On the 11th day, in situ working heart-brainstem preparations were made and the activity of thoracic sympathetic nerve (SNA) was recorded. Five sequential activations of peripheral chemoreceptors (0.05% KCN, 50 μl, i.a.) were performed before and after either bilateral ADM (100 fmol/100 nl) or saline injections (100 nl) into the RVLM. We observed that the combination of ADM injections and peripheral chemoreflex stimulations evoked LTF of SNA only in CIH but not in controls rats (140±47%, vs. control: 35±23% of baseline at 60 min, p<0.05). There was no LTF of SNA in CIH or control rats that were submitted to multiple peripheral chemoreflex activation with saline injected into RVLM. These results demonstrate that in rats submitted to CIH, ADM associated with multiple chemoreflex activation is involved in the development of LTF of SNA. These findings may explain, at least in part, the sympathetic overactivity observed in CIH rats.

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.

To investigate the contribution of central vasopressin receptors to blood pressure (BP) and heart rate (HR) response to stress we injected non-peptide selective V1a (SR49059), V1b (SSR149415), V2 (SR121463) receptor antagonists, diazepam or vehicle in the lateral cerebral ventricle of conscious freely moving rats stressed by blowing air on their heads for 2 min. Cardiovascular effects of stress were evaluated by analyzing maximum increase of BP and HR (MAX), latency of maximum response (LAT), integral under BP and HR curve (∫), duration of their recovery and spectral parameters of BP and HR indicative of increased sympathetic outflow (LFBP and LF/HFHR). Moreover, the increase of serum corticosterone was measured. Exposure to air-jet stress induced simultaneous increase in BP and HR followed by gradual decline during recovery while LFBP oscillation remained increased as well as serum corticosterone level. Rats pre-treated with vasopressin receptor antagonists were not sedated while diazepam induced sedation that persisted during exposure to stress. V1a, V1b and V2 receptor antagonists applied separately did not modify basal values of cardiovascular parameters but prevented the increase in ∫BP. In addition, V1b and V2 receptor antagonists reduced BPMAX whereas V1a, V1b antagonist and diazepam reduced HRMAX induced by exposure to air-jet stress. All drugs shortened the recovery period, prevented the increase of LFBP without affecting the increase in serum corticosterone levels. Results indicate that vasopressin receptors located within the central nervous system mediate, in part, the cardiovascular response to air-jet stress without affecting either the neuroendocrine component or inducing sedation. They support the view that the V1b receptor antagonist may be of potential therapeutic value in reducing arterial pressure induced by stress-related disorders.

The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca(2+) imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion.

Mutations in the arginine vasopressin (AVP)-neurophysin II (NP-II) gene that affect the folding and transport of the prohormone result in loss of secretion of the anti-diuretic hormone AVP from pituitary nerve terminals and cause autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). One such mutation consists of the replacement of a Cys residue at position 98 with a stop codon (C98X) in the AVP precursor (corresponding to C67X in NP domain). In neuroblastoma cells over-expressing this truncated AVP precursor autophagy, a macromolecular degradation process, was shown to be essential for assuring cell survival. In the present study, we investigated the role of the Akt pro-survival signalling in the regulation of autophagy and of apoptosis linked with the handling of C98X AVP. Impairing autophagy-lysosomal sequestration or cathepsin D (CD)-mediated proteolysis triggered the activation of the intrinsic death pathway of apoptosis in C98X-expressing cells, but not in the wild-type -AVP-expressing cells. This was shown by the expression of a Vps34 dominant negative, which down-regulates the PI3k class III-dependent signalling needed for autophagosome (APH) formation, by genetic silencing as a result of RNA interference (RNAi) of Lamp2, a protein indispensable for the fusion of APHs with lysosomes, and by RNAi silencing of the lysosomal protease CD. Ectopic expression of either the wild-type or the mutated C98X AVP altered neither the expression nor the phosphorylation of the pro-survival signalling molecule Akt. Strikingly, the ectopic adenoviral-directed expression of a constitutively active Akt, instead of preserving cell survival, resulted in the suppression of autophagy, and precipitated Bax-mediated cell death. The present data demonstrate the need for autophagy-mediated degradation of mutated C98X peptides, which otherwise become toxic to the cell, and suggest that, in the presence of mis-folded proteins, the stimulation of the Akt signalling counteracts the beneficial effects of autophagy and precipitates cell death. It follows that growth factors impinging on the Akt pathway may have deleterious effect in neurones expressing mutant neuropeptides. This can provide an explanation for the late onset and progressive neuronal cell loss observed in hypothalamic magnocellular neurones of adFNDI patients.

We have used microarrays to comprehensively describe the transcriptomes of the supraoptic nucleus (SON), the paraventricular nucleus (PVN) and the neurointermediate lobe of adult male Sprague‐Dawley (SD) and Wistar‐Kyoto (WKY) rats, as well as the PVN of Wistar rats. Comparison of these gene lists has enabled us to identify surprisingly large differences in hypothalamic‐neurohypophyseal system gene expression patterns in these three strains. We have also shown that different transcript populations are enriched in the PVN and the SON of SD and WKY rats. The transcriptome differences catalogued here may be molecular substrates for the neuro‐humoral phenotypic differences exhibited by different strains of rats.