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Lipopolysaccharide (LPS) is often used to mimic acute infection and induces hypophagia, the selective partitioning of fat for energy, and fever. Interleukin-10 (IL-10) is an anti-inflammatory cytokine expressed in the brain which attenuates LPS-induced hypophagia; however the potential sites of interaction within the brain have not been investigated. Hypothalamic orexin (ORX) and melanin-concentrating hormone (MCH) regulate energy expenditure and food intake although the regulation of these neuropeptides through the interactions between central IL-10 and the inflammatory consequences of peripheral LPS have not been investigated. The present study in the rat investigated during the dark phase of the light-dark cycle the ability of central IL-10 (250 ng, i.c.v.) to attenuate the changes in food intake, energy substrate partitioning, and central Fos expression within the hypothalamus to peripheral LPS (100 microg/kg, i.p.); Fos expression changes specifically within ORX and MCH neurons were also investigated. Central IL-10 attenuated the peripheral LPS-induced hypophagia, reduction in motor activity, fever and reduction in respiratory exchange ratio. Central IL-10 also attenuated peripheral LPS-induced increases in Fos expression within ORX neurons and decreases in Fos expression within unidentified cells of the caudal arcuate nucleus. In contrast, both IL-10 and LPS injection independently decreased Fos expression within MCH neurons. The present study provides further insight into the interactions within the brain between the anti-inflammatory cytokine IL-10, the inflammatory consequences of LPS, and neuropeptides known to regulate energy expenditure and food intake., (Copyright 2010 Elsevier Ltd. All rights reserved.)

ANG II, the main circulating effector hormone of the renin-angiotensin system, is produced by enzymatic cleavage of angiotensinogen. The present study aimed to examine whether targeted deletion of the angiotensinogen gene (Agt) altered brain ANG II receptor density or responsiveness to ANG II. In vitro autoradiography was used to examine the distribution and density of angiotensin type 1 (AT(1)) and type 2 receptors. In most brain regions, the distribution and density of angiotensin receptors were similar in brains of Agt knockout mice (Agt(-/-)) and wild-type mice. In Agt(-/-) mice, a small increase in AT(1) receptor binding was observed in the rostral ventrolateral medulla (RVLM), a region that plays a critical role in blood pressure regulation. To examine whether Agt(-/-) mice showed altered responses to ANG II, blood pressure responses to intravenous injection (0.01-0.1 microg/kg) or RVLM microinjection (50 pmol in 50 nl) of ANG II were recorded in anesthetized Agt(-/-) and wild-type mice. Intravenous injections of phenylephrine (4 microg/kg and 2 microg/kg) were also made in both groups. The magnitude of the pressor response to intravenous injections of ANG II or phenylephrine was not different between Agt(-/-) and wild-type mice. Microinjection of ANG II into the RVLM induced a pressor response, which was significantly smaller in Agt(-/-) compared with wild-type mice (+10 + or - 1 vs. +23 + or - 4 mmHg, respectively, P = 0.004). Microinjection of glutamate into the RVLM (100 pmol in 10 nl) produced a robust pressor response, which was not different between Agt(-/-) and wild-type mice. A diminished response to ANG II microinjection in the RVLM of Agt(-/-) mice, despite an increased density of AT(1) receptors suggests that signal transduction pathways may be altered in RVLM neurons of Agt(-/-) mice, resulting in attenuated cellular excitation.

Splicing is a post-transcriptional modification of RNA during which introns are removed and exons are joined. Most of the mammalian genes undergo constitutive and alternative splicing events. In addition to the strong signals of the splice sites, splicing is influenced at a distance by a range of trans factors that interact with cis regulatory elements and influence the spliceosome. The intention of the present mini-review is to give some insights into the complexity of this interaction and to introduce the consequences of some kinds of detrimental genetic variation on alternative splicing and disease.

Brown adipose tissue (BAT), body and brain temperatures, as well as behavioral activity, arterial pressure and heart rate, increase episodically during the waking (dark) phase of the circadian cycle in rats. Phase-linking of combinations of these ultradian (<24 h) events has previously been noted, but no synthesis of their overall interrelationships has emerged. We hypothesized that they are coordinated by brain central command, and that BAT thermogenesis, itself controlled by the brain, contributes to increases in brain and body temperature. We used chronically implanted instruments to measure combinations of bat, brain and body temperatures, behavioral activity, tail artery blood flow, and arterial pressure and heart rate, in conscious freely moving Sprague-Dawley rats during the 12-h dark active period. Ambient temperature was kept constant for any particular 24-h day, varying between 22 and 27 degrees C on different days. Increases in BAT temperature (> or = 0.5 degrees C) occurred in an irregular episodic manner every 94+/-43 min (mean+/-SD). Varying the temperature over a wider range (18-30 degrees C) on different days did not change the periodicity, and neither body nor brain temperature fell before BAT temperature episodic increases. These increases are thus unlikely to reflect thermoregulatory homeostasis. Episodic BAT thermogenesis still occurred in food-deprived rats. Behavioral activity, arterial pressure (18+/-5 mmHg every 98+/-49 min) and heart rate (86+/-31 beats/min) increased approximately 3 min before each increase in BAT temperature. Increases in BAT temperature (1.1+/-0.4 degrees C) were larger than corresponding increases in brain (0.8+/-0.4 degrees C) and body (0.6+/-0.3 degrees C) temperature and the BAT episodes commenced 2-3 min before body and brain episodes, suggesting that BAT thermogenesis warms body and brain. Hippocampal 5-8 Hz theta rhythm, indicating active engagement with the environment, increased before the behavioral and autonomic events, suggesting coordination by brain central command as part of the 1-2 h ultradian basic rest-activity cycle (BRAC) proposed by Kleitman.

RFamide-related peptide-3 (RFRP-3) is a neuropeptide produced in cells of the paraventricular nucleus and dorsomedial nucleus of the ovine hypothalamus. In the present study, we show that these cells project to cells in regions of the hypothalamus involved in energy balance and reproduction. A retrograde tracer (FluoroGold) was injected into either the arcuate nucleus, the lateral hypothalamic area or the ventromedial nucleus. The distribution and number of retrogradely-labelled RFRP-3 neurones was determined. RFRP-3 neurones projected to the lateral hypothalamic area and, to a lesser degree, to the ventromedial nucleus and the arcuate nucleus. Double-label immunohistochemistry was employed to identify cells receiving putative RFRP-3 input to cells in these target regions. RFRP-3 cells were seen to project to neuropeptide Y and pro-opiomelanocortin neurones in the arcuate nucleus, orexin and melanin-concentrating hormone neurones in the lateral hypothalamic area, as well as orexin cells in the dorsomedial nucleus and corticotrophin-releasing hormone and oxytocin cells in the paraventricular nucleus. Neurones expressing gonadotrophin-releasing hormone in the preoptic area were also seen to receive input from RFRP-3 projections. We conclude that RFRP-3 neurones project to hypothalamic regions and cells involved in regulation of energy balance and reproduction in the ovine brain.

Central neural control of complex feeding behaviour is likely to be influenced by a number of factors including homeostatic responses to peripheral nutrient status, cortical integration of feeding-related cues and the underlying reward value of food. We have used retrogradely transported neurotropic viruses, as tools to map chains of synaptically-connected neurons, in conjunction with neurochemical markers of feeding-related peptides to expand the blueprint of the circuitries that underlie these different components of feeding behaviour. We have identified projections to insular and anterior cingulate cortex, extending from the arcuate nucleus through synaptic relays in the lateral hypothalamic area and midline thalamic nuclei. Cortically projecting neurons from the hypothalamic arcuate nucleus were found predominantly in its lateral aspects and contained anorexigenic peptides with no representation amongst more medially-positioned neurons containing orexigenic peptides. Largely overlapping pathways were shown to project multisynaptically to the shell of the nucleus accumbens but those with origins in the arcuate nucleus had either orexigenic or anorexigenic phenotypes. Similar to the cortical projections, those relaying to the nucleus accumbens in the lateral hypothalamus contained the orexigenic peptides orexin-A and melanin-concentrating hormone in approximately 30% of cases. Common to the neural pathways directed to all three virally-injected areas were nodes of synaptic relays in the lateral hypothalamus and midline thalamic nuclei. These regions are well positioned to integrate sensory information about energy homeostasis and the reward value of food in the passage of this information to the 'ingestive cortex'.

Numerous studies have demonstrated that administration of rimonabant (SR 141716), a CB(1) receptor antagonist, causes a decrease in energy intake. However, the mechanisms by which rimonabant exerts its anorectic actions are unclear. The main focus of the study reported here was to establish the chemical identity of neurons that may subserve the anorectic effects of rimonabant. As such three approaches were utilised: (i) the identification of rimonabant-activated neurons using Fos as a marker of neuronal activity; (ii) the identification of the chemical phenotype of rimonabant-activated neurons by combining immunocytochemical identification of Fos and feeding-related peptides; and (iii) the evaluation of the effect of rimonabant on messenger RNA (mRNA) and protein for a number of feeding-related peptides. Rimonabant-induced Fos-positive nuclei were localized within a range of discrete hypothalamic regions with a predominance in the parvocellular part of the paraventricular nucleus of the hypothalamus, dorsomedial hypothalamus, arcuate nucleus and lateral hypothalamic area. Furthermore, Fos labelling within these hypothalamic regions was colocalized with anorexigenic and orexigenic peptides including melanin-concentrating hormone (MCH), orexin, cocaine- and amphetamine-regulated transcript (CART) and alpha-melanocyte-stimulating hormone (alpha-MSH). Rimonabant specifically induced a decrease in NPY and an increase in CART and alpha-MSH mRNA and protein, consistent with its effect in reducing food intake and increasing energy expenditure. As such these data provide insights into the mechanisms of action that may underpin rimonabant's effects on energy balance and body weight.

Background: Bariatric surgery is currently the only anti-obesity therapy that can deliver weight loss of up to 20-30% of body weight. Laparoscopic adjustable gastric banding (LAGB) and Roux-en-y gastric bypass are the most commonly performed of these surgeries. The mechanisms by which LAGB initiates an increase in satiety remain completely unknown. The aim of this study is to establish a rodent model of adjustable gastric banding (AGB) that will enable investigation of these mechanisms., Methods: Sprague-Dawley rats were implanted with adjustable gastric bands immediately below the gastro-esophageal junction around the glandular stomach. This band, as in humans, can be inflated via an exteriorized port resulting in an incremental impact on the stomach., Results: Rats with an incremental inflation of the AGB showed a clear stepwise reduction in food intake and body weight. Normal food intake and body weight gain were restored with band deflation. Barium-assisted X-ray of the stomach showed the formation of a small gastric pouch proximal to the inflated band in a manner analogous to the human LAGB., Conclusions: This is the first animal model of the AGB that allows incremental inflation for optimal tightening of the band in the conscious animal with corresponding effects on food intake and body weight. This model will allow measurement of acute and chronic neural and hormonal changes following activation of the band in the conscious animal and will provide the potential to inform and improve surgical approaches that are at the forefront of obesity treatments.

The cannabinoid CB1 receptor antagonist rimonabant (SR 141716) produces a sustained decrease in body weight on a background of a transient reduction in food intake. An increase in energy expenditure has been implicated, possibly mediated via peripheral endocannabinoid system; however, the role of the central endocannabinoid system is unclear. The present study investigates this role. Rimonabant (10 mg/kg IP) was administered for 21 days to rats surgically implanted with biotelemetry devices to measure temperature in the interscapular brown adipose tissue (BAT). BAT temperature as a putative measure of thermogenesis in the BAT, physical activity, body weight, food intake, as well as changes in UCP1 messenger RNA (mRNA) and protein were measured. In addition, role of the CNS in mediating these actions of rimonabant was determined in rats where the BAT was sympathetically denervated. As expected, chronic administration of rimonabant significantly reduced body weight for the entire treatment period despite only a transient decrease in food intake. There was a profound increase in BAT temperature, particularly during the dark phase of each circadian cycle throughout the treatment period. A corresponding increase in uncoupling protein (UCP1) was also observed following chronic rimonabant treatment. The rimonabant-induced elevation in BAT temperature and decrease in body weight were significantly attenuated following denervation, indicating an involvement of the CNS. These findings suggest that the long-term weight loss associated with rimonabant treatment is due at least in part to an elevation in energy expenditure, represented here by elevated temperature recorded in the BAT, which is mediated primarily by the central endocannabinoid system.

The paraventricular nucleus (PVN) and the periventricular nucleus (Pe) are important neuroendocrine centers, but the neuronal input to these regions is poorly defined in nonrodent species. We utilized the retrograde transport of injected tracers to determine the neural input to these two nuclei in the ovine brain. Adult Corriedale ewes were studied following FluoroGold injection into either the PVN (n = 5) or the Pe (n = 3). Both the PVN and the Pe were found to receive neuronal input from a number of hypothalamic nuclei. Projections to the PVN from the lateral hypothalamic area were from neurons that produce melanin-concentrating hormone or orexins and a subset of those from the arcuate nucleus were immunopositive for neuropeptide Y and gamma-melanocyte stimulating hormone. This pathway was verified by staining of terminals in the PVN. Input to the PVN from the brain stem was seen to originate from the catecholaminergic and serotoninergic neurons. The projections to the PVN and Pe from hypothalamic and brain stem regions in the sheep brain are generally similar to those in the rat, with some minor differences. These studies highlight the differences in the afferent input to these two closely related nuclei in the ovine brain., (2009 S. Karger AG, Basel.)

The administration of antipsychotic drugs to human patients or experimental animals leads to significant weight gain, which is widely presumed to be driven by hyperphagia; however, the contribution from energy expenditure remains unclear. These studies aim to examine the contribution of shifts in energy expenditure, particularly those involving centrally mediated changes in thermogenesis, to the body weight gain associated with the administration of olanzapine to female Sprague Dawley rats. Olanzapine (6 mg/kg/day orally) caused a transient increase in food intake but a maintained increase in body weight. When pair-fed rats were treated with olanzapine, body weight continued to rise compared to vehicle-treated rats, consistent with a reduction in energy expenditure. Brown adipose tissue (BAT) temperature, measured using biotelemetry devices, decreased immediately after the onset of olanzapine treatment and remained depressed, as did physical activity. UCP1 expression in interscapular BAT was reduced following chronic olanzapine treatment. An acute injection of olanzapine was preceded by an injection of a retrograde tracer into the spinal cord to evaluate the nature of the olanzapine-activated neural pathway. Levels of Fos protein in a number of spinally projecting neurons within discrete hypothalamic and brainstem sites were elevated in olanzapine-treated rats. Some of these neurons in the perifornical region of the lateral hypothalamus (LHA) were also Orexin A positive. These data collectively show a significant impact of thermogenesis (and physical activity) on the weight gain associated with olanzapine treatment. The anatomical studies provide an insight into the central neuroanatomical substrate that may subserve the altered thermogenic responses brought about by olanzapine.

Water intakes in response to hypertonic, hypovolemic, and dehydrational stimuli were investigated in mice lacking angiotensin II as a result of deletion of the angiotensinogen gene (Agt-/- mice), and in C57BL6 wild-type (WT) mice. Baseline daily water intake in Agt-/- mice was approximately threefold that of WT mice because of a renal developmental disorder of the urinary concentrating mechanisms in Agt-/- mice. Intraperitoneal injection of hypertonic saline (0.4 and 0.8 mol/l NaCl) caused a similar dose-dependent increase in water intake in both Agt-/- and WT mice during the hour following injection. As well, Agt-/- mice drank appropriate volumes of water following water deprivation for 7 h. However, Agt-/- mice did not increase water or 0.3 mol/l NaCl intake in the 8 h following administration of a hypovolemic stimulus (30% polyethylene glycol sc), whereas WT mice increased intakes of both solutions during this time. Osmoregulatory regions of the brain [hypothalamic paraventricular and supraoptic nuclei, median preoptic nucleus, organum vasculosum of the lamina terminalis (OVLT), and subfornical organ] showed an increased number of neurons exhibiting Fos-immunoreactivity in response to intraperitoneal hypertonic NaCl in both Agt-/- mice and WT mice. Polyethylene glycol treatment increased Fos-immunoreactivity in the subfornical organ, OVLT, and supraoptic nuclei in WT mice but only increased Fos-immunoreactivity in the supraoptic nucleus in Agt-/- mice. These data show that brain angiotensin is not essential for the adequate functioning of neural pathways mediating osmoregulatory thirst. However, angiotensin II of either peripheral or central origin is probably necessary for thirst and salt appetite that results from hypovolemia.

The pathways involved in the emotional aspects of thirst, the arousal and affect associated with the generation of thirst and the motivation to obtain satiation, have been studied but remain poorly understood. Rats were therefore injected with the neurotropic virus pseudorabies in either the insular or cingulate cortex. After 2 days of infection, pseudorabies-positive neurons were identified within the thalamus and lamina terminalis. In a separate group of rats, the retrograde tracer cholera toxin subunit b (CTb) was used in combination with either isotonic (0.15 M NaCl) or hypertonic (0.8 M NaCl) saline (1 ml/100 g body wt ip). Rats injected with CTb in the insular cortex and stimulated with hypertonic saline had increased numbers of Fos/CTb double-positive neurons in the paraventricular, rhomboid, and reuniens thalamic nuclei, whereas those rats injected with CTb in the cingulate cortex and challenged with hypertonic saline had increased numbers of Fos/CTb double-positive neurons in the medial part of the mediodorsal, interanteromedial, anteromedial, and ventrolateral part of the laterodorsal thalamic nuclei. Rats injected with CTb in the dorsal midline of the thalamus and challenged with hypertonic saline had increased numbers of Fos/CTb double-positive neurons within the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and insular cortex but not the subfornical organ. A small proportion of the CTb-positive neurons in the OVLT were immunopositive for transient receptor potential vanilloid 1, a putative osmoresponsive membrane protein. These results identify functional thalamocortical pathways involved in relaying osmotic signals to the insular and cingulate cortex and may provide a neuroanatomical framework for the emotional aspects of thirst.

The ventromedial nucleus of the hypothalamus (VMN) and the arcuate nucleus (ARC) are two centres regulating energy balance and food intake, but inter-connectivity of these nuclei is not well defined in non-rodent species. In this study, we performed retrograde tracing and immunohistochemistry in the ovine brain with ewes receiving FluoroGold (FG) injections into either ARC or VMN for the mapping of retrogradely labelled cells. Strong reciprocal connections were found between the two regions. The distribution of the FG labelled neurons in other regions of the hypothalamus and brain stem was also mapped. Some of the cells projecting from ARC to VMN were immunopositive for neuropeptide Y, galanin, adrenocorticotropin (marker of pro-opiomelanocortin cells) or tyrosine hydroxylase (marker of dopaminergic cells). Melanin-concentrating hormone and orexin neurons in the lateral hypothalamic area were also found to provide input to the VMN and ARC. This observed interconnectivity between regions important for metabolic regulation and other neuroendocrine functions presumably allows coordinated functions. Input to both the ARC and VMN from other brain regions, such as brain stem cell groups, provides a further level of regulation. These data provide a substrate upon which further understanding of appetite regulation and neuroendocrine function can be derived in this species., ((c) 2007 S. Karger AG, Basel)

The concept of 'command neurons', whereby single neurons mediate complex and complementary motor functions to generate a stereotyped behaviour, is well developed in invertebrate physiology. The term has also been adopted more recently to explain the neural basis of 'fight or flight'. In this study we have investigated the possibility that single lateral hypothalamic neurons have the necessary neuroanatomical connections to coordinate two complementary limbs of body weight control, feeding and thermogenesis, thereby acting as 'command neurons'. The transynaptic retrograde transport of pseudorabies virus (Bartha) from a thermogenic endpoint in the brown adipose tissue of rats has been used in conjunction with other neuronal tracers, introduced into putative CNS feeding centres, to assess the potential for the involvement of command neurons in coordinating these processes. In discrete regions of the lateral hypothalamus, neurons have been identified which have the necessary complement of orexigenic peptides and collateral branching axons to both putative feeding sites and thermogenic sites in brown fat to qualify as candidate central command neurons controlling body weight.

The recently identified neuropeptide QRFP(26) is predominantly expressed in the hypothalamus and was suggested to play a role in the regulation of food intake following the observation of an acute orexigenic effect after central administration in mice. QRFP(26) exerts its effect via GPR103 and a newly identified receptor in mouse. The aim of our study was (a) to investigate the distribution of QRFP(26) and a newly discovered QRFP receptor mRNA in rat and (b) to further characterize the effects of central administration of QRFP(26) on energy balance in rats. QRFP(26) mRNA was detected in the retrochiasmatic nucleus, periventricular nucleus, arcuate nucleus and restricted areas of the lateral nucleus of the hypothalamus. We found an additional receptor with high homology for GPR103 in rat. This receptor increases inositol triphosphate production in transfected cells in presence of QRFP(26) and its mRNA was particularly enriched in ventral and posterior thalamic groups, anterior hypothalamus and medulla. When QRFP(26) (10 microg and 50 microg) was administered centrally before the start of the light phase both doses increased food intake for 2 h after injection without reaching statistical significance. QRFP(26) caused no changes in locomotor activity or energy expenditure. In summary, central QRFP(26) injection causes slight and transient hyperphagia in rats without changing any other energy balance parameters after 24 h. We conclude that QRFP(26) has limited impact on the central regulation of energy balance in rats and that its essential function remains to be clarified.

Thirst and resultant water drinking can arise in response to deficits in both the intracellular and extracellular fluid compartments. Inhibitory influences mediating the satiation of thirst also are necessary to prevent overhydration. The brain regions that underpin the generation or inhibition of thirst in these circumstances can be categorized as sensory, integrative, or cortical effector sites. The anterior cingulate cortex and insula are activated in thirsty human beings as shown by functional brain-imaging techniques. It is postulated that these sites may be cortical effector regions for thirst. A major sensory site for generating thirst is the lamina terminalis in the forebrain. Osmoreceptors within the organum vasculosum of the lamina terminalis and subfornical organ detect systemic hypertonicity. The subfornical organ mediates the dipsogenic actions of circulating angiotensin II and relaxin. Major integrative sites are the nucleus of the tractus solitarius, the lateral parabrachial nucleus, the midbrain raphé nuclei, the median preoptic nucleus, and the septum. Despite these advances, most of the neural pathways and neurochemical mechanisms subserving the genesis of thirst remain to be elucidated.

Leptin, a peptide hormone normally associated with body weight homeostasis, is implicated in the generation of obesity-induced hypertension. Administration of leptin increases sympathetic nerve activity and blood pressure; however, the neural circuity involved in this pressor effect is not clearly defined. In this review we describe experiments in which pseudorabies virus was injected into the heart, kidney and the vasculature within skeletal muscle to reveal the distribution of neurones in the hypothalamus that project to these cardiovascular tissues. This distribution is compared to the well-documented distribution of leptin receptors. Finally we discuss microinjection studies designed to examine the effect of leptin, in these regions, on sympathetic nerve discharge and arterial blood pressure. Leptin injected directly into the ventromedial hypothalamus, arcuate nucleus and lateral hypothalamic area (particularly the perifornical area) increased lumbar sympathetic nerve activity. In addition, microinjection into the ventromedial hypothalamus and parvocellular paraventricular nucleus increased blood pressure. Our results demonstrate a discrete set of hypothalamic pathways that may underlie the involvement of leptin in obesity-induced hypertension.

Thirst motivates animals to seek fluid and drink it. It is regulated by the central nervous system and arises from neural and chemical signals from the periphery interacting in the brain to stimulate a drive to drink. Our research has focussed on the lamina terminalis and the manner in which osmotic and hormonal stimuli from the circulation are detected by neurons in this region and how that information is integrated with other neural signals to generate thirst. Our studies of osmoregulatory drinking in the sheep and rat have produced evidence that osmoreceptors for thirst exist in the dorsal cap of the organum vasculosum of the lamina terminalis (OVLT) and in the periphery of the subfornical organ, and possibly also in the median preoptic nucleus. In the rat, the hormones angiotensin II and relaxin act on neurons in the periphery of the subfornical organ to stimulate drinking. Studies of human thirst using functional magnetic resonance imaging (fMRI) techniques show that systemic hypertonicity activates the lamina terminalis and the anterior cingulate cortex, but the neural circuitry that connects sensors in the lamina terminalis to cortical regions subserving thirst remains to be determined. Regarding pathophysiological influences on thirst mechanisms, both excessive (polydipsia) and inadequate (hypodisia) water intake may have dire consequences. One of the most common primary polydipsias is that observed in some cases of schizophrenia. The neural mechanisms causing the excessive water intake in this disorder are unknown, so too are the factors that result in impaired thirst and inadequate fluid intake in some elderly humans.

The lamina terminalis, located in the anterior wall of the third ventricle, is comprised of the subfornical organ, median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT). The subfornical organ and OVLT are two of the brain's circumventricular organs that lack the blood-brain barrier, and are therefore exposed to the ionic and hormonal environment of the systemic circulation. Previous investigations in sheep and rats show that this region of the brain has a crucial role in osmoregulatory vasopressin secretion and thirst. The effects of lesions of the lamina terminalis, studies of immediate-early gene expression and electrophysiological data show that all three regions of the lamina terminalis are involved in osmoregulation. There is considerable evidence that physiological osmoreceptors subserving vasopressin release are located in the dorsal cap region of the OVLT and possibly also around the periphery of the subfornical organ and in the MnPO. The circulating peptide hormones angiotensin II and relaxin also have access to peptide specific receptors (AT(1) and LGR7 receptors, respectively) in the subfornical organ and OVLT, and both angiotensin II and relaxin act on the subfornical organ to stimulate water drinking in the rat. Studies that combined neuroanatomical tracing and detection of c-fos expression in response to angiotensin II or relaxin suggest that both of these circulating peptides act on neurones within the dorsal cap of the OVLT and the periphery of the subfornical organ to stimulate vasopressin release.

The aim of this study was to determine, in conscious rats, whether elevated concentrations of circulating angiotensin II activate neurones in both the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) that project to the bed nucleus of the stria terminalis (BNST). The strategy employed was to colocalize retrogradely transported cholera toxin B subunit (CTB) from the BNST, with elevated levels of Fos protein in response to angiotensin II. Circulating angiotensin II concentrations were increased by either intravenous infusion of angiotensin II or subcutaneous injection of isoproterenol. Neurones exhibiting Fos in response to angiotensin II were present in the subfornical organ, predominantly in its central core but with some also seen in its peripheral aspect, the dorsal and lateral margins of the OVLT, the supraoptic nucleus and the parvo- and magnocellular divisions of the paraventricular nucleus. Fos-labelling was not apparent in control rats infused with isotonic saline intravenously or injected with either CTB or CTB conjugated to gold particles (CTB-gold) only. Of the neurones in the subfornical organ that were shown by retrograde labelling to project to BNST, approximately 50% expressed Fos in response to isoproterenol. This stimulus also increased Fos in 33% of neurones in the OVLT that project to BNST. Double-labelled neurones were concentrated in the central core of the subfornical organ and lateral margins of the OVLT in response to increased circulating angiotensin II resulting from isoproterenol treatment. These data support a role for circulating angiotensin II acting either directly or indirectly on neurones in subfornical organ and OVLT that project to the BNST and provide further evidence of functional regionalization within the subfornical organ and the OVLT. The function of these pathways is yet to be determined; however, a role in body fluid homeostasis is possible.

The influence of urocortin (UCN) on ingestive behaviours and brain neural activity, as measured immunohistochemically by the presence of Fos protein, was determined in mice. Rat UCN was administered by continuous intracerebroventricular (ICV) or subcutaneous (SC) infusion. ICV infusion of UCN (100 ng/h, 14 days) transiently reduced daily food and water intakes (days 1-4) but body weight was reduced from day 2 into the post-infusion period. Sodium intake was reduced from day 3 to the end of infusion. SC infusion of UCN caused similar but smaller reductions in food and water intakes and body weight, without change in sodium intake. In separate experiments, Fos immunoreactivity was increased in several brain nuclei known to be involved in the control of body fluid and energy homeostasis, e.g. central nucleus of the amygdala, median preoptic nucleus, bed nucleus of the stria terminalis and arcuate nucleus. Increased Fos expression was similar for ICV and SC infusions when measured on days 2-3 or 6-7 of infusion. In conclusion, increases of brain activity by UCN may be associated with stimulation of adrenocorticotrophic hormone release and sympathetic nervous activity, but increases may also indicate suppression of ingestive behaviours by stimulating central inhibitory mechanisms located in areas known to control body fluid and energy homeostasis.

Angiotensinogen, the precursor molecule for angiotensins I, II and III, and the enzymes renin, angiotensin-converting enzyme (ACE), and aminopeptidases A and N may all be synthesised within the brain. Angiotensin (Ang) AT(1), AT(2) and AT(4) receptors are also plentiful in the brain. AT(1) receptors are found in several brain regions, such as the hypothalamic paraventricular and supraoptic nuclei, the lamina terminalis, lateral parabrachial nucleus, ventrolateral medulla and nucleus of the solitary tract (NTS), which are known to have roles in the regulation of the cardiovascular system and/or body fluid and electrolyte balance. Immunohistochemical and neuropharmacological studies suggest that angiotensinergic neural pathways utilise Ang II and/or Ang III as a neurotransmitter or neuromodulator in the aforementioned brain regions. Angiotensinogen is synthesised predominantly in astrocytes, but the processes by which Ang II is generated or incorporated in neurons for utilisation as a neurotransmitter is unknown. Centrally administered AT(1) receptor antagonists or angiotensinogen antisense oligonucleotides inhibit sympathetic activity and reduce arterial blood pressure in certain physiological or pathophysiological conditions, as well as disrupting water drinking and sodium appetite, vasopressin secretion, sodium excretion, renin release and thermoregulation. The AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and plays a role in memory mechanisms. In conclusion, angiotensinergic neural pathways and angiotensin peptides are important in neural function and may have important homeostatic roles, particularly related to cardiovascular function, osmoregulation and thermoregulation.

The brain's three sensory circumventricular organs, the subfornical organ, organum vasculosum of the lamina terminalis and the area postrema lack a blood brain barrier and are the only regions in the brain in which neurons are exposed to the chemical environment of the systemic circulation. Therefore they are ideally placed to monitor the changes in osmotic, ionic and hormonal composition of the blood. This book describes their. General structure and relationship to the cerebral ventricles Regional subdivisions Vasculature and barrier properties Neurons, glia and ependymal cells Receptors, neurotransmitters, neuropeptides and enzymes Neuroanatomical connections Functions.

We have used a transgenic approach to visualize functionally activated neurons and their projections. The transgenic mice contain a tau-lacZ fusion gene regulated by the promoter for c-fos, an immediate early gene that is rapidly induced in neurons after functional stimulation. Constitutive expression of beta-galactosidase (beta-gal), the lacZ product, was low and in accord with previous reports of c-fos expression. However, expression of beta-gal in positive neurons was clearly in cell bodies, axons, and dendrites. Treatment of the mice with kainic acid, a strong inducer of c-fos expression, resulted in high induction of beta-gal. beta-gal was induced in the same defined populations of neurons in the brain as those that express c-fos after kainic acid induction. Furthermore, the pattern of beta-gal expression within the neurons changed over time after kainic acid treatment. Early after kainate treatment, beta-gal was found mainly in cell bodies; at later times, expression extended further along the neuronal processes. This expression pattern is consistent with induction and anterograde transport of the Fos-Tau-beta-gal protein in the neurons. To test whether a functionally activated pathway could be visualized, transgenic mice were deprived of water, which activates nuclei involved in body fluid homeostasis. beta-gal induction was traced in neurons and their processes in the lamina terminalis, in magnocellular neurons of the supraoptic and paraventricular nuclei, and in their projections to the posterior pituitary gland. This strategy allowed the mapping of an activated osmoregulatory pathway. This transgenic approach may have general application in the mapping of functionally activated circuitry in the brain.

Relaxin, a peptide hormone secreted by the corpus luteum during pregnancy, exerts actions on reproductive tissues such as the pubic symphysis, uterus, and cervix. It may also influence body fluid balance by actions on the brain to stimulate thirst and vasopressin secretion. We mapped the sites in the brain that are activated by i.v. infusion of a dipsogenic dose of relaxin (25 microg/h) by immunohistochemically detecting Fos expression. Relaxin administration resulted in increased Fos expression in the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and magnocellular neurons in the supraoptic and paraventricular nuclei. Ablation of the SFO abolished relaxin-induced water drinking, but did not prevent increased Fos expression in the OVLT, supraoptic or paraventricular nuclei. Although ablation of the OVLT did not inhibit relaxin-induced drinking, it did cause a large reduction in Fos expression in the supraoptic nucleus and posterior magnocellular subdivision of the paraventricular nucleus. In vitro single-unit recording of electrical activity of neurons in isolated slices of the SFO showed that relaxin (10(-7) M) added to the perfusion medium caused marked and prolonged increase in neuronal activity. Most of these neurons also responded to 10(-7) M angiotensin II. The data indicate that blood-borne relaxin can directly stimulate neurons in the SFO to initiate water drinking. It is likely that circulating relaxin also stimulates neurons in the OVLT that influence vasopressin secretion. These two circumventricular organs that lack a blood-brain barrier may have regulatory influences on fluid balance during pregnancy in rats.

The identification of leptin and a range of novel anorectic and orexigenic peptides has focussed attention on the neural circuitry involved in the genesis of food intake and the reflex control of thermogenesis. Here, the neurotropic virus pseudorabies has been utilised in conjunction with the immunocytochemical localisation of a variety of neuroactive peptides and receptors to better define the pathways in the rat hypothalamus directed polysynaptically to the major thermogenic endpoint, brown adipose tissue. Infected neurones were detected initially in the stellate ganglion, then in the spinal cord followed by the appearance of third-order premotor neurones in the brainstem and hypothalamus. Within the hypothalamus these were present in the paraventricular nucleus, lateral hypothalamus, perifornical region, and retrochiasmatic nucleus. At slightly longer survival times virus-infected neurones appeared in the arcuate nucleus and dorsomedial hypothalamus. Neurones in the retrochiasmatic nucleus and in the adjacent lateral arcuate nucleus which project to the brown adipose tissue express cocaine- and amphetamine-regulated transcript, pro-opiomelanocortin and leptin receptors. Neurones in the lateral hypothalamus, a site traditionally associated with the promotion of feeding, project to brown adipose tissue and large numbers of these contained melanin-concentrating hormone and orexin A and B. These data provide part of an anatomical framework which subserves the regulation of energy expenditure.

Neurotropic viruses have been used over the last 10 years to map the distribution of chains of synaptically connected neurons in the CNS. The peptide content of infected neurons has been determined in a number of cases immunohistochemically. However, it has been unclear whether specific mRNA can be assessed in virus-infected neurons. We have established a technique which enables the identification of viral protein and mRNA in the same neuron. In the present study pseudorabies virus retrogradely transported from the kidney was localised using immunohistochemistry and mRNA for the angiotensin II AT(1A) receptor was detected by hybridisation histochemistry. Virus protein was visualised using an immunohistochemical procedure with diaminobenzidine as the chromogen and the same sections were exposed to radioactively labelled ((35)S) riboprobes, hybridising the angiotensin II AT(1A) receptor. The combination of these two approaches resulted in the identification of neurons shown to project polysynaptically to the kidney and express AT(1A) mRNA. These data provide neuroanatomical support for previous physiological observations that ablation of the lamina terminalis and administration of losartan, the AT(1) receptor antagonist, blocks the inhibition of renal sympathetic nerve activity following centrally injected Ang II in rats and sheep [5].

This study was undertaken to determine if neurons in the lamina terminalis, previously identified as projecting to the kidney (35), were responsive to alterations in stimuli associated with fluid balance homeostasis. Neurons in the lamina terminalis projecting to the kidney were identified by the retrograde transynaptic transport of Bartha's strain of pseudorabies virus in anesthetized rats. Rats were also exposed to 24-h water deprivation, intravenous hypertonic saline, or intracerebroventricular ANG II. To determine if "kidney-directed" neurons were activated following each stimulus, brain sections that included the lamina terminalis were examined immunohistochemically for viral antigen and Fos protein. With the exception of ANG II in the subfornical organ, all regions of the lamina terminalis contained neurons that were significantly activated by water deprivation, hypertonic saline, and ANG II. These results provide evidence for a neural substrate, which may underpin some of the effects of hypertonic saline and ANG II on renal function thought to be mediated through the lamina terminalis.

Angiotensinogen, the precursor molecule of the peptides angiotensin I, II, and III, is synthesized in the brain and the liver. Evidence is reviewed that angiotensin II, and possibly angiotensin III, that are generated within the brain act within neural circuits of the central nervous system to regulate body fluid balance. Immunohistochemical studies in the rat brain have provided evidence of angiotensin-containing neurons, especially in the hypothalamic paraventricular nucleus, subfornical organ, periventricular region, and nucleus of the solitary tract, as well as in extensive angiotensin-containing fiber pathways. Angiotensin immunoreactivity is observed by electron microscope in synaptic vesicles in several brain regions, the most prominent of these being the central nucleus of the amygdala. Neurons in many parts of the brain (lamina terminalis, paraventricular and parabrachial nuclei, ventrolateral medulla, and nucleus of the solitary tract) known to be involved in the regulation of body fluid homeostasis exhibit angiotensin receptors of the AT(1) subtype. Pharmacological studies in several species show that intracerebroventricular administration of AT(1) receptor antagonist drugs inhibit homeostatic responses to the central administration of hypertonic saline, intravenous infusion of the hormone relaxin, or thermal dehydration. Responses affected by centrally administered AT(1) antagonists are water drinking, vasopressin secretion, natriuresis, increased arterial pressure, reduced renal renin release, salt hunger, and thermoregulatory adjustments. We conclude that angiotensinergic neural pathways in the brain probably have an important homeostatic function, especially in regard to osmoregulation and thermoregulation, and the maintenance of arterial pressure.

The ovarian hormone relaxin, in addition to its role in pregnancy, exerts an action on the brain to influence oxytocin and vasopressin secretion, water drinking, and cardiovascular function. Intravenous (i.v.) infusion of relaxin causes an acute water drinking response, confirming its role as a dipsogenic hormone. The aim of this study was to determine whether neurones in the lamina terminalis, which project to the hypothalamic paraventricular and supraoptic nuclei, are activated by elevated levels of circulating relaxin in conscious rats. Immunocytochemistry combined with retrograde neuronal tracing with cholera toxin B subunit conjugated to cholera toxin B (CTB-gold) was used to identify populations of neurones responding with elevated cells of Fos protein to i.v. relaxin administration and which project to these specific hypothalamic sites. Neurones exhibiting Fos were present in the outer parts of the subfornical organ (SFO), the dorsal part of the organum vasculosum (OVLT), the supraoptic nucleus and the paraventricular nucleus. These did not occur in control rats with i.v. infusions of isotonic saline. Approximately 90% of neurones concentrated in the outer parts of the SFO and in the dorsal OVLT showed both retrogradely transported CTB-gold and Fos in response to i.v. infusion of relaxin. These data support a role for relaxin acting on the brain to regulate body fluid and electrolyte homeostasis by activating neural pathways subserving water drinking, vasopressin and oxytocin secretion.

The retrograde transynaptic transport of pseudorabies virus was used in conjunction with hybridisation histochemistry for the angiotensin II AT1A receptor, to characterise neurons in the lamina terminalis projecting to the kidney. These data demonstrate that some neurons in the lamina terminalis, that project polysynaptically to the kidney, may be responsive to angiotensin II.

Angiotensin II acts within the hypothalamic paraventricular nucleus (PVN) to help mediate a number of autonomic and endocrine responses. Evidence is sparse in regard to the particular neuronal cell groups that exhibit angiotensin II type 1 receptors within the PVN, and does not exist in relation to specified efferent neuronal populations in the nucleus. In the present experiments, retrogradely transported neuronal tracers were utilized in conjunction with immunohistochemistry using a well characterized polyclonal antibody raised against a decapeptide sequence at the carboxy terminus of the AT1 receptor, to determine whether it is preferentially distributed amongst different efferent populations within the PVN. The AT1 receptor is not associated with neurones in the PVN that project axons to the spinal cord, dorsomedial or ventrolateral medulla but coexists strongly with neurones in the anterior parvocellular division of the nucleus which direct axons to the median eminence. Such neurones often contain corticotropin releasing factor. These findings highlight the role that angiotensin II and AT1 receptors in the PVN may play in the mediation of responses to stress.

Neural activity, as measured immunohistochemically by the presence of Fos protein, was determined in the lamina terminalis, a thin strip of tissue forming the anterior wall of the third brain ventricle, after adrenalectomy. Several weeks after surgery, the adrenalectomized rats were maintained with access to water and a low sodium diet for five days. In addition, hypertonic (0.5M) NaCl solution was available for the entire five-day period (sodium available) or only during the first four days (sodium unavailable). The number of neurons expressing Fos, determined at the end of the fifth day, was increased in the adrenalectomized rats with or without NaCl solution to drink. Fos activity in the median preoptic nucleus was increased only in adrenalectomized rats without access to NaCl solution. Treatment of adrenalectomized rats with the sodium-retaining mineralocorticoid hormone, deoxycorticosterone, at the end of the fourth day, decreased Fos expression in the subfornical organ and the organum vasculosum of the lamina terminalis when NaCl solution was available but not when the NaCl solution was unavailable. In the adrenalectomized rats with NaCl solution available, mineralocorticoid treatment decreased both urinary sodium excretion and daily sodium intake. Brain nuclei in the lamina terminalis also became activated in intact rats made sodium deplete by treatment with the diuretic, furosemide. Relative to sodium-deplete intact rats, however, sodium-deplete adrenalectomized rats had a greater number of neurons expressing Fos in the organum vasculosum. Treatment of sodium-deplete rats, adrenalectomized or intact, with the angiotensin II-type 1 receptor antagonist, ZD7155, decreased sodium intake and Fos expression in the subfornical organ but not in the organum vasculosum of the lamina terminalis or median preoptic nucleus. In conclusion, the results demonstrated that activation of the brain nuclei located in the lamina terminalis of adrenalectomized rats was primarily related to sodium deficit and not to the absence of the mineralocorticoid hormones, although the adrenal hormones may have a role in limiting the activation of organum vasculosum of the lamina terminalis during sodium depletion. Furthermore, the results obtained with the administration of the angiotensin receptor antagonist are consistent with the proposal that sodium appetite of the sodium-deplete rat, adrenalectomized or intact, is mediated by circulating angiotensin II acting in the subfornical organ.

These data demonstrate a chain of synaptically connected neurons extending to the rat kidney through several levels of the neuraxis from the forebrain, and the lamina terminalis -- an area known to be involved in the regulation of body fluid homeostasis. The Bartha strain of pseudorabies virus was injected into the kidney of male Sprague-Dawley rats, resulting in retrograde infections in spinal cord segments (T1-T8), and successive infection in five autonomic 'premotor' areas of the brain, the rostroventrolateral medulla, rostroventromedial medulla, raphe nuclei, A5 region of the pons, and the paraventricular nucleus of the hypothalamus, as well as the nucleus of the solitary tract, locus coeruleus, and subcoeruleus nuclei. Higher order labelling was found in regions of the forebrain, including the organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, medial preoptic area, bed nucleus of the stria terminalis, anteroventral periventricular nucleus, lateral preoptic area, suprachiasmatic nucleus, retrochiasmatic nucleus, primary motor cortex, and visceral cortex. This polysynaptic pathway to the kidney may form the substrate underlying the impact of forebrain structures on renal function.

The lamina terminalis, which forms most of the anterior wall of the third ventricle, consists of the median preoptic nucleus and two circumventricular organs (CVOs), the subfornical organ and organum vasculosum of the lamina terminalis. These latter two regions lack a blood-brain barrier and, unlike other regions of the brain, are influenced by the hormonal and ionic composition of the blood. The CVOs of the lamina terminalis are rich in receptors for a number of circulating peptides and the subfornical organ and the OVLT are clearly established as the prime cerebral targets for circulating angiotensin II, atrial natriuretic peptide (AVP) and relaxin to influence central nervous system pathways regulating body fluid homeostasis. Together with the median preoptic nucleus, these two CVOs also detect changes and relay neural signals relating to the tonicity of body fluids and play important roles in osmoregulatory fluid intake and excretion. The neural circuitry of the lamina terminalis involves both afferent and efferent connections to several other regions of the brain, and neurons within the individual components of lamina terminalis are reciprocally connected with each other. This neural circuitry subserves the influence that the lamina terminalis exerts on vasopressin secretion, thirst, the appetite for salt, renal sodium excretion and renin secretion by the kidney. Copyright 1999 Harcourt Publishers Ltd.

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.(J Histochem Cytochem 47:507-515, 1999)

Unlike circulating steroid hormones, which have a relatively unhindered passage into the central nervous system, blood-borne peptides are usually restricted by the blood-brain barrier. Some circulating peptides, such as angiotensin II, atrial natriuretic peptide and relaxin, influence central neural pathways subserving cardiovascular and body fluid homeostasis by acting on neurons in the subfornical organ, organum vasculosum of the lamina terminalis and area postrema, all of which lack a blood-brain barrier. There are some circulating peptides such as insulin and leptin that are transported from the bloodstream across cerebral blood vessel walls into sites in the hypothalamus that have appropriate neural connections to influence food intake and sympathetic control of brown fat.

Neurones situated in the lamina terminalis (organum vasculosum of the lamina terminalis, median preoptic nucleus and subfornical organ) as well as within medial and lateral parts of the preoptic area and in the insular cortex become transneuronally labelled following pseudorabies virus injections into the submandibular or the sublingual gland. These neurones are efferently connected to a chain of central neurones directed to secretory or vascular tissue of the submandibular or the sublingual gland. By varying the postinoculation time a stepwise infection of different forebrain nuclei was registered, with the hypothalamic paraventricular nucleus and the lateral hypothalamic area being the first forebrain structures labelled. Such early infected neurones within these hypothalamic nuclei are in all likelihood third order neurones regulating salivary secretion and might have functioned as relays transmitting virus to other forebrain structures. The above mentioned forebrain areas together with several other hypothalamic nuclei as well as the bed nucleus of the stria terminalis, the central nucleus of the amygdala and the substantia innominata, seem to be the widespread anatomical basis for the central regulation of salivary gland function., (Copyright 1998 Elsevier Science B.V.)

The effect of intracerebroventricular (ICV) injections of synthetic human or rat relaxin (25 or 250 ng) on the distribution of Fos detected immunohistochemically in the rat forebrain was investigated. Following ICV relaxin, many Fos-positive neurons were observed in the periphery of the subfornical organ, dorsal part of the organum vasculosum of the lamina terminalis, throughout the median preoptic nucleus, supraoptic nucleus and hypothalamic paraventricular nucleus. Such effects did not occur following ICV injection of artificial cerebrospinal fluid or the separated A and B chains of relaxin, nor following the intravenous injection of 250 ng of relaxin. Both vasopressin and oxytocin containing neurons identified immunohistochemically in the supraoptic and paraventricular nuclei exhibited Fos following ICV relaxin, and many neurons in the medial parvocellular part of the paraventricular nucleus contained Fos. The results indicate that centrally administered relaxin may increase neuronal activity in regions of the hypothalamus and lamina terminalis which are associated with cardiovascular and body fluid regulation and oxytocin secretion.

Bradykinin B2 receptors were localized in the sheep brain and spinal cord by quantitative in vitro autoradiography using a radiolabelled and specific bradykinin B2 receptor antagonist analogue, 3-4-hydroxyphenyl-propionyl-D-Arg0-[Hyp3,Thi5,D-Tic 7,Oic8]bradykinin, (HPP-HOE 140). This radioligand displays high affinity and specificity for bradykinin B2 receptors. The respective K(i) values of 0.32, 1.37 and 156 nM were obtained for bradykinin, HOE140 and D-Arg[Hyp3,D-Phe7,Leu8]bradykinin competing for radioligand binding to lamina II of sheep spinal cord sections. Using this radioligand, we have demonstrated the distribution of bradykinin B2 receptors in many brain regions which have not been previously reported. The highest density of bradykinin B2 receptors occur in the pleoglial periaqueductal gray, oculomotor and trochlear nuclei and the circumventricular organs. Moderate densities of receptors occur in the substantia nigra, particularly the reticular part, the posterior thalamic and subthalamic nuclei, zona incerta, the red and pontine nuclei, some of the pretectal nuclei and in discrete layers of the superior colliculus. In the hindbrain, moderate levels of bradykinin B2 receptor binding occur in the nucleus of the solitary tract, and in spinal trigeminal, inferior olivary, cuneate and vestibular nuclei. Laminae II, X and dorsal root ganglia display the most striking binding densities in the spinal cord, while the remainder of the dorsal and ventral horn display a low and diffuse density of binding. Bradykinin B2 receptors are extensively distributed throughout the sheep brain and spinal cord, not only to sensory areas but also to areas involved in motor activity.

Immunohistochemical techniques were used to detect Fos in the brain following subcutaneous administration of the angiotensin converting enzyme inhibitors captopril or enalapril at 0.5 mg/kg to conscious rats. Increased Fos-like immunoreactivity was observed in many neurons in the lamina terminalis, and in regions of the hypothalamus. Captopril at this dose also caused water drinking in other rats. Pre-treatment with the angiotensin AT1 receptor antagonist ZD7155 (10 mg/kg) given subcutaneously prevented the captopril-induced increase in Fos in the lamina terminalis. This dose of ZD7155 also prevented captopril-induced drinking in other rats. With a higher dose (50 mg/kg) of captopril or enalapril, there was no increase in Fos in the lamina terminalis. This dose of captopril was not dipsogenic. The results are consistent with the proposal that the lower dose (0.5 mg/kg) of captopril or enalapril increases circulating angiotensin I levels which are then converted to angiotensin II in the organum vasculosum of the lamina terminalis and subfornical organ. Stimulation of neurons at these sites may subserve water drinking and sodium appetite.

1. Autoradiographic binding studies have shown that the AT(1) receptor is the predominant angiotensin II (AngII) receptor subtype in the central nervous system (CNS). Major sites of AT(1) receptors are the lamina terminalis, hypothalamic paraventricular nucleus, the lateral parabrachial nucleus, rostral and caudal ventrolateral medulla, nucleus of the solitary tract and the intermediolateral cell column of the thoraco-lumbar spinal cord. 2. While there are differences between species, AT(2) receptors are found mainly in the cerebellum, inferior olive and locus coeruleus of the rat. 3. Circulating AngII acts on AT(1) receptors in the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) to stimulate neurons that may have a role in initiating water drinking. 4. Centrally administered AngII may act on AT(1) receptors in the median preoptic nucleus and elsewhere to induce drinking, sodium appetite, a sympathetic vasoconstrictor response and vasopressin secretion. 5. Recent evidence shows that centrally administered AT(1) antagonists inhibit dipsogenic, natriuretic, pressor and vasopressin secretory responses to intracerebroventricular infusion of hypertonic saline. This suggests that an angiotensinergic neural pathway has a role in osmoregulatory responses. 6. Central angiotensinergic pathways which include neural inputs to the rostral ventrolateral medulla may use AT(1) receptors and play a role in the function of sympathetic pathways maintaining arterial pressure., (1996 Blackwell Publishing Asia Pty Ltd.)

The aims of this study are twofold. The first is to describe the ultrastructural morphology of putative osmoreceptors concentrated in the ventral aspect of the lamina terminalis in the rat forebrain. The second is to determine whether or not these neurons lie within an area which lacks a blood-brain barrier, i.e. the organum vasculosum lamina terminalis. The results describe a compact population of neurons in the ventral part of the lamina terminalis which both respond to an osmotic challenge and project directly to the supraoptic nucleus. Injection of horseradish peroxidase into the circulation, as a marker to define areas of the brain without a blood-brain barrier, indicates that these neurons are in the dorsal aspect of the organum vasculosum of the lamina terminalis. An ultrastructural analysis of the neurons in this area, which respond to an osmotic challenge with an elevation of Fos protein, show them to have no specific morphological characteristics which differentiate them from other, non-responsive neurons in the organum vasculosum of the lamina terminalis. However, one possible exception is that osmotically sensitive neurons have a less indented nucleus, suggesting that they are in a more active state than their non-osmotically sensitive neighbours. It is concluded that neurons in this region of the brain are candidate structures for the "receptors" which mediate vasopressin release in response to an osmotic challenge. The response of only a subset of neurons in the organum vasculosum of the lamina terminalis to an osmotic stimulus, despite an apparent morphological homogeneity and the ability of blood borne agents to reach all parts of the structure suggests that osmoresponsiveness is conferred by unique membrane properties or intracellular processing events. The presence of synaptic input to osmoresponsive cells indicates a potential for integration of other inputs at this level.

In the sheep spinal cord, a high density of [125I]angiotensin IV binding sites was localized to the perikaryon and processes of all somatic motor neurons, the autonomic motor neurons in the lateral horns of thoracic and lumbar segments and all dorsal root ganglia, but was low in lamina II of all dorsal horns. At supraspinal levels, [125I]angiotensin IV binding was abundant in numerous motor associated regions, with weaker binding observed in the sensory regions. This wide distribution pattern suggests an important role for the binding site in the central nervous system.

Fos immunoreactivity in the rat brain after intracerebroventricular (ICV) angiotensin II (ANG II) was compared with that induced by intravenous ANG II. ANG II was infused into the lateral ventricle (at 1 ng/min) or femoral vein (at 5 micrograms/h) of conscious rats. After 90 min, rats were killed and Fos was detected by immunohistochemistry. Both infusions caused Fos immunoreactivity to be present in the lamina terminalis, hypothalamic supraoptic, and paraventricular nuclei, bed nucleus of the stria terminalis, and central amygdaloid nucleus. However, distributions of Fos immunoreactivity within the lamina terminalis differed with the different routes of infusion. Intravenous ANG II caused intense Fos immunoreactivity mainly in the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT). By contrast, ICV ANG II caused intense Fos immunoreactivity predominantly in the median preoptic nucleus and juxtaventricular neurons of the SFO and OVLT. These results suggest that IV ANG II induces behavioural and endocrine responses by direct actions on the SFO and OVLT, whereas ICV ANG II directly stimulates neurons in the median preoptic nucleus as well neurons in the SFO and OVLT.

The beta adrenergic agonist isoproterenol has been used in these studies to elevate circulating levels of angiotensin II. Neurons in the brain responsive to the subcutaneous infusion of isoproterenol were identified using an antibody to Fos, the protein product of c-fos which is now used extensively as a marker of activated neurons. Fos-positive neurons were present in a range of specific forebrain and hind brain regions. Infusion of losartan (an angiotensin II type receptor antagonist) showed that neurons in the lamina terminalis were activated directly or indirectly by angiotensin II, whereas other neurons in the hypothalamus and brain stem were responsive as a consequence of the peripheral vasodilation caused by isoproterenol. The distribution of activated neurons in the lamina terminalis was consistent with that of neurons thought to be involved in water drinking.

A population of depressor neurons in the caudal ventrolateral medulla that project to the rostral ventrolateral medulla may mediate the baroreceptor reflex. The aim of the present study was to determine the anatomical distribution of the population of neurons in the caudal ventrolateral medulla that mediate the baroreceptor reflex. Injection of the retrogradely transported tracer, rhodamine-labelled latex beads, into the pressor area of the rostral ventrolateral medulla of rats was used to identify neurons in the caudal ventrolateral medulla with projections to that area. Barosensitive neurons were identified by immunohistochemical detection of the protein Fos, a marker of neuronal activation, following infusion of the pressor agent phenylephrine (10 micrograms/kg/min, i.v. for 2 h n = 5). Isotonic saline was infused into control animals (n = 4). Neurons in the caudal ventrolateral medulla with projections to the rostral ventrolateral medulla were located at all rostrocaudal levels examined between 1 mm caudal and 0.4 mm rostral of the obex. Compared to saline infused rats, phenylephrine infusion induced a significant increase in the proportion of those neurons that expressed Fos (14% vs. 1% P < 0.000.1). These barosensitive neurons were found mainly at the level of the obex, between the lateral reticular nucleus and the nucleus ambiguus. In conclusion, this study is the first to show the distribution of the population of barosensitive neurons in the caudal ventrolateral medulla that project to the pressor region of the rostroventrolateral medulla. The results suggest there is a subpopulation of depressor neurons, confined to a small region of the rostral part of the caudal ventrolateral medulla, that are likely to be the interneurons that mediate the baroreceptor-reflex response.