Charles Hindmarch

A comprehensive description of the transcriptome of the hypothalamoneurohypophyseal system in euhydrated and dehydrated rats

The hypothalamoneurohypophyseal system (HNS) consists of the large peptidergic magnocellular neurons of the supraoptic hypo thalamic nucleus (SON) and the paraventricular hypothalamic nucleus (PVN), the axons of which course through the internal zone of the median eminence and terminate at blood capillaries of the posterior lobe of the pituitary gland. The HNS is a specialized brain neurosecretory apparatus responsible for the production of the antidiuretic peptide hormone vasopressin (VP). VP maintains water balance by promoting water conservation at the level of the kidney. Dehydration evokes a massive increase in the regulated release of VP from magnocellular neuron axon terminals in the posterior pituitary, which is accompanied by a plethora of changes in the morphology, electrophysiological properties, and biosynthetic and secretory activity of the HNS. We wish to understand this functional plasticity in terms of the differential expression of genes. We have therefore used microarrays to comprehensively catalog the genes expressed in the PVN, the SON and the neurointermediate lobe of the pituitary gland of control and dehydrated rats. Comparison of these gene lists has enabled us to identify transcripts that are regulated as a consequence of dehydration as well as RNAs that are enriched in the PVN or the SON. We suggest that these differentially expressed genes represent candidate regulators and effectors of HNS activity and remodeling.

Microarray Analysis of the Transcriptome of the Subfornical Organ in the Rat: Regulation by Fluid and Food Deprivation

We have employed microarray technology using Affymetrix 230 2.0 genome chips to initially catalog the transcriptome of the subfornical organ (SFO) under control conditions and to also evaluate the changes (common and differential) in gene expression induced by the challenges of fluid and food deprivation. We have identified a total of 17,293 genes tagged as present in one of our three experimental conditions, transcripts, which were then used as the basis for further filtering and statistical analysis. In total, the expression of 46 genes was changed in the SFO following dehydration compared with control animals (22 upregulated and 24 downregulated), with the largest change being the greater than fivefold increase in brain-derived neurotrophic factor (BDNF) expression, while significant changes in the expression of the calcium-sensing (upregulated) and apelin (downregulated) receptors were also reported. In contrast, food deprivation caused greater than twofold changes in a total of 687 transcripts (222 upregulated and 465 downregulated), including significant reductions in vasopressin, oxytocin, promelanin concentrating hormone, cocaine amphetamine-related transcript (CART), and the endothelin type B receptor, as well as increases in the expression of the GABA(B) receptor. Of these regulated transcripts, we identified 37 that are commonly regulated by fasting and dehydration, nine that were uniquely regulated by dehydration, and 650 that are uniquely regulated by fasting. We also found five transcripts that were differentially regulated by fasting and dehydration including BDNF and CART. In these studies we have for the first time described the transcriptome of the rat SFO and have in addition identified genes, the expression of which is significantly modified by either water or food deprivation.

Antagonism of orexin receptors significantly lowers blood pressure in spontaneously hypertensive rats

•  Orexin is involved in blood pressure regulation. Certain forms of stress or central administration of orexin increases blood pressure and sympathetic nerve activity in normal rats; orexin knockout mice and orexin neuron‐ablated transgenic rats have lower basal blood pressure. The role of orexin in hypertension remains unknown. •  RT‐PCR shows a strong trend towards an increased orexin‐A mRNA expression in the rostral ventrolateral medulla in adult spontaneously hypertensive rats. •  In adult spontaneously hypertensive rats, blocking orexin receptors by oral administration of an antagonist, almorexant, significantly lowers blood pressure in wakefulness and non‐rapid eye movement sleep during dark and light cycles, an effect accompanied by decreased sympathetic vasomotor tone and noradrenaline levels in cerebrospinal fluid and plasma. •  Antagonizing orexin receptors had no effect on resting blood pressure in normal rats. •  The orexin system participates in the pathogenesis of high blood pressure in spontaneously hypertensive rats. Modulation of the orexin system could be a potential target in treating some forms of hypertension.

G protein-coupled receptors in the hypothalamic paraventricular and supraoptic nuclei - serpentine gateways to neuroendocrine homeostasis

Graphical abstract Highlights ► The paraventricular and supraoptic nuclei of the hypothalamus are regulators of homeostasis. ► Over one hundred G protein-coupled receptors are expressed in each of these nuclei. ► The receptors have many functions including modulating neuropeptide synthesis and release. ► 20–30% of the receptors are ‘orphans’ whose endogenous ligand and function is unknown.

Excessive leukotriene B4 in nucleus tractus solitarii is prohypertensive in spontaneously hypertensive rats.

Inflammation within the brain stem microvasculature has been associated with chronic cardiovascular diseases. We found that the expression of several enzymes involved in arachidonic acid-leukotriene B4 (LTB4) production was altered in nucleus tractus solitarii (NTS) of spontaneously hypertensive rat (SHR). LTB4 produced from arachidonic acid by 5-lipoxygenase is a potent chemoattractant of leukocytes. Leukotriene B4-12-hydroxydehydrogenase (LTB4-12-HD), which degrades LTB4, was downregulated in SHR rats compared with that in Wistar-Kyoto rats. Quantitative real-time PCR revealed that LTB4-12-HD was reduced by 63% and 58% in the NTS of adult SHR and prehypertensive SHR, respectively, compared with that in age-matched Wistar-Kyoto rats (n=6). 5-lipoxygenase gene expression was upregulated in the NTS of SHR (≈50%; n=6). LTB4 levels were increased in the NTS of the SHR, (17%; n=10, P<0.05). LTB4 receptors BLT1 (but not BLT2) were expressed on astroglia in the NTS but not neurons or vessels. Microinjection of LTB4 into the NTS of Wistar-Kyoto rats increased both leukocyte adherence and arterial pressure for over 4 days (peak: +15 mm Hg; P<0.01). In contrast, blockade of NTS BLT1 receptors lowered blood pressure in the SHR (peak: −13 mm Hg; P<0.05) but not in Wistar-Kyoto rats. Thus, excessive amounts of LTB4 in NTS of SHR, possibly as a result of upregulation of 5-lipoxygenase and downregulation of LTB4-12-HD, can induce inflammation. Because blockade of NTS BLT1 receptors lowered arterial pressure in the SHR, their endogenous activity may contribute to the hypertensive state of this rodent model. Thus, inflammatory reactions in the brain stem are causally associated with neurogenic hypertension.

The hypothalamic-neurohypophyseal system: from genome to physiology

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.

Transcription Factor Expression in the Hypothalamo-Neurohypophyseal System of the Dehydrated Rat: Upregulation of Gonadotrophin Inducible Transcription Factor 1 mRNA Is Mediated by cAMP-Dependent Protein Kinase A

The supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamo-neurohypophyseal system (HNS) undergo a dramatic function-related plasticity during dehydration. We hypothesize that alterations in steady-state transcript levels might be partially responsible for this remodeling. In turn, regulation of transcript abundance might be mediated by transcription factors. We used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation in the SON. We observed downregulation of 10 and upregulation of 28 transcription factor transcripts. For five of the upregulated mRNAs, namely gonadotropin inducible ovarian transcription factor 1 (Giot1), Giot2, cAMP-responsive element binding protein 3-like 1, CCAAT/enhancer binding protein β, and activating transcription factor 4, in situ hybridization was used to confirm the array results, demonstrating a significant increase in expression in SON and PVN magnocellular neurons (MCNs) after 3 d of water deprivation and, in some cases, upregulation in parvocellular PVN neurons. Using a viral vector expressing a potent inhibitor of cAMP-dependent protein kinase A (PKA), we show that the osmotically induced increase in the abundance of transcripts encoding Giot1 is mediated in vivo by the PKA pathway. We thus suggest that signaling pathways activated by dehydration in MCNs mediate transcription factor gene activation, which, in turn, regulate target genes that mediate HNS remodeling.

A comparison of physiological and transcriptome responses to water deprivation and salt loading in the rat supraoptic nucleus

Salt loading (SL) and water deprivation (WD) are experimental challenges that are often used to study the osmotic circuitry of the brain. Central to this circuit is the supraoptic nucleus (SON) of the hypothalamus, which is responsible for the biosynthesis of the hormones, arginine vasopressin (AVP) and oxytocin (OXT), and their transport to terminals that reside in the posterior lobe of the pituitary. On osmotic challenge evoked by a change in blood volume or osmolality, the SON undergoes a function-related plasticity that creates an environment that allows for an appropriate hormone response. Here, we have described the impact of SL and WD compared with euhydrated (EU) controls in terms of drinking and eating behavior, body weight, and recorded physiological data including circulating hormone data and plasma and urine osmolality. We have also used microarrays to profile the transcriptome of the SON following SL and remined data from the SON that describes the transcriptome response to WD. From a list of 2,783 commonly regulated transcripts, we selected 20 genes for validation by qPCR. All of the 9 genes that have already been described as expressed or regulated in the SON by osmotic stimuli were confirmed in our models. Of the 11 novel genes, 5 were successfully validated while 6 were false discoveries.

Switching control of sympathetic activity from forebrain to hindbrain in chronic dehydration

Non‐technical summary  Dehydration, a life‐threatening condition, occurs when the body does not replace adequate water lost through urination, sweating or when ill with diarrhoea. This presents the body with a major challenge of maintaining blood pressure – essential for consciousness that is dependent on the degree of body hydration, which dictates blood volume. We know that a major control mechanism involves a brain region called the hypothalamus that automatically maintains blood pressure. Our study has described the gene networks in key brain regions involved in the response to dehydration. We reveal a new structure in the brain that regulates blood pressure in dehydration and a unique genetic mechanism that exists within it. Moreover, our study unearths a remarkable form of flexibility within the brain during dehydration that involves switching control of blood pressure between two spatially distinct structures. We have provided new mechanistic insight to explain how the brain maintains body stability in face of the significant challenge of low water content.

Inhibition of Egr1 expression underlies the anti-mitogenic effects of cAMP in vascular smooth muscle cells

Aims Cyclic AMP inhibits vascular smooth muscle cell (VSMC) proliferation which is important in the aetiology of numerous vascular diseases. The anti-mitogenic properties of cAMP in VSMC are dependent on activation of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), but the mechanisms are unclear. Methods and results Selective agonists of PKA and EPAC synergistically inhibited Egr1 expression, which was essential for VSMC proliferation. Forskolin, adenosine, A2B receptor agonist BAY60-6583 and Cicaprost also inhibited Egr1 expression in VSMC but not in endothelial cells. Inhibition of Egr1 by cAMP was independent of cAMP response element binding protein (CREB) activity but dependent on inhibition of serum response element (SRE) activity. SRF binding to the Egr1 promoter was not modulated by cAMP stimulation. However, Egr1 expression was dependent on the SRF co-factors Elk1 and 4 but independent of MAL. Inhibition of SRE-dependent Egr1 expression was due to synergistic inhibition of Rac1 activity by PKA and EPAC, resulting in rapid cytoskeleton remodelling and nuclear export of ERK1/2. This was associated with de-phosphorylation of the SRF co-factor Elk1. Conclusion cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression. This occurs, at least in part, via inhibition of Rac1 activity leading to rapid actin-cytoskeleton remodelling, nuclear export of ERK1/2, impaired Elk1-phosphorylation and inhibition of SRE activity. This identifies one of the earliest mechanisms underlying the anti-mitogenic effects of cAMP in VSMC but not in endothelial cells, making it an attractive target for selective inhibition of VSMC proliferation.