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

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

Adrenocortical sensitivity, moderated by ongoing stress, predicts drinking intensity in alcohol-dependent men.

Allostatic load from both environmental stressors and persistent glucocorticoid secretion has been associated with disease severity in alcohol dependence. Heightened relapse risk and/or drinking severity, in particular, may be a reaction to alcohol- and withdrawal-induced changes in physiological stress response systems coupled with ongoing life stress, although their shared contributions upon drinking severity have not been assessed. To investigate the combined contribution of hypothalamic-pituitary-adrenal (HPA) reactivity and environmental stressors (e.g., ongoing life stress) to relapse severity in alcohol-dependent men following treatment, plasma adrenocorticotropin (ACTH) and cortisol were obtained in 4-6 weeks abstinent alcohol-dependent men (n=41) following a psychosocial stressor [the Trier Social Stress Test (TSST)] and two pharmacological provocations [ovine corticotropin releasing factor (oCRH) and cosyntropin]. Following treatment discharge, drinking outcomes (primary outcome: drinks per drinking day (DDD); secondary outcomes: total drinks and drinking days) were assessed weekly and ongoing life stress was assessed biweekly for 24 weeks following treatment discharge. Generalized estimating equation models of drinking severity were fit with basal and stimulated ACTH and cortisol concentrations as predictors and ongoing life stress as the moderator. Greater levels of life stress were independently associated with greater drinking intensity (DDD and total drinks) but not frequency (days drinking). Higher basal cortisol:ACTH or provoked cortisol:ACTH ratios were strongly associated with greater post-treatment DDD in individuals who experienced higher levels of ongoing stress. In conclusion, ongoing life stress is associated with post-treatment drinking intensity in alcohol dependent men; stress also strengthens the relationship between adrenocortical sensitivity and post-treatment drinking. Physiological measures of allostatic load and environmental stressors conjointly increase relapse intensity.

Circadian and homeostatic modulation of functional connectivity and regional cerebral blood flow in humans under normal entrained conditions.

Diurnal rhythms have been observed in human behaviors as diverse as sleep, olfaction, and learning. Despite its potential impact, time of day is rarely considered when brain responses are studied by neuroimaging techniques. To address this issue, we explicitly examined the effects of circadian and homeostatic regulation on functional connectivity (FC) and regional cerebral blood flow (rCBF) in healthy human volunteers, using whole-brain resting-state functional magnetic resonance imaging (rs-fMRI) and arterial spin labeling (ASL). In common with many circadian studies, we collected salivary cortisol to represent the normal circadian activity and functioning of the hypothalamic-pituitary-adrenal (HPA) axis. Intriguingly, the changes in FC and rCBF we observed indicated fundamental decreases in the functional integration of the default mode network (DMN) moving from morning to afternoon. Within the anterior cingulate cortex (ACC), our results indicate that morning cortisol levels are negatively correlated with rCBF. We hypothesize that the homeostatic mechanisms of the HPA axis has a role in modulating the functional integrity of the DMN (specifically, the ACC), and for the purposes of using fMRI as a tool to measure changes in disease processes or in response to treatment, we demonstrate that time of the day is important when interpreting resting-state data.

Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis.

The neurohypophysis is a crucial component of the hypothalamo-pituitary axis, serving as the site of release of hypothalamic neurohormones into a plexus of hypophyseal capillaries. The growth of hypothalamic axons and capillaries to the forming neurohypophysis in embryogenesis is therefore crucial to future adult homeostasis. Using ex vivo analyses in chick and in vivo analyses in mutant and transgenic zebrafish, we show that Fgf10 and Fgf3 secreted from the forming neurohypophysis exert direct guidance effects on hypothalamic neurosecretory axons. Simultaneously, they promote hypophyseal vascularisation, exerting early direct effects on endothelial cells that are subsequently complemented by indirect effects. Together, our studies suggest a model for the integrated neurohemal wiring of the hypothalamo-neurohypophyseal axis.

The cortisol awakening response in context.

The cortisol awakening response (CAR) is a crucial point of reference within the healthy cortisol circadian rhythm, with cortisol secretion typically peaking between 30 and 45 min post awakening. This chapter reviews the history of investigation into the CAR and highlights evidence that its regulation is relatively distinct from cortisol secretion across the rest of the day. It is initiated by awakening, under the influence of the hypothalamic suprachiasmatic nucleus, and "fine tuned" by a direct neural input to the adrenal cortex by the sympathetic nervous system. This chapter also examples the CAR in relation to other awakening-induced processes, such as restoration of consciousness, attainment of full alertness, changes in other hormones, changes in the balance of the immune system, and mobilization of the motor system, and speculates that there is a role for the CAR in these processes.

Gene expression of vascular endothelial growth factor-A in the pituitary during formation of the vascular system in the hypothalamic-pituitary axis of the rat.

Techniques involving fluorescein-5-isothiocyanate-conjugated gelatin injection, immunohistochemistry, and in situ reverse transcription/polymerase chain reaction (RT-PCR) revealed a close relationship between vascular endothelial growth factor (VEGF)-A-expressing cells and microvessels in the hypothalamic-pituitary axis of the rat. In situ RT-PCR clearly indicated the presence of VEGF-A mRNA-expressing cells in the pars tuberalis and in the pars distalis both at embryonic day 15.5 (E15.5) and in later developmental stages. The primary capillaries extended along the developing pars tuberalis, whereas the portal vessels penetrated into the pars distalis at E15.5 and subsequently expanded into the lobe to connect with the secondary capillary plexus, emerging in the pars distalis. At the same time, several VEGF-A-positive cells appeared in the pars distalis. These VEGF-A-positive cells were found to correspond to a portion of adrenocorticotropin (ACTH) cells by dual-staining for in situ RT-PCR and immunohistochemistry, suggesting that some ACTH cells have the potential to produce VEGF-A. Thus, the present study suggests that VEGF-A is involved in the development of the primary capillaries and in the vascularization of the pars distalis, but not in the portal vessels since the formation of portal vessels begins at E13.5, before the appearance of VEGF-A in the rostral region of the pars distalis.

Hormonal cycle modulates arousal circuitry in women using functional magnetic resonance imaging.

Sex-specific behaviors are in part based on hormonal regulation of brain physiology. This functional magnetic resonance imaging (fMRI) study demonstrated significant differences in activation of hypothalamic-pituitary-adrenal (HPA) circuitry in adult women with attenuation during ovulation and increased activation during early follicular phase. Twelve normal premenopausal women were scanned twice during the early follicular menstrual cycle phase compared with late follicular/midcycle, using negative valence/high arousal versus neutral visual stimuli, validated by concomitant electrodermal activity (EDA). Significantly greater magnitude of blood oxygenation level-dependent signal changes were found during early follicular compared with midcycle timing in central amygdala, paraventricular and ventromedial hypothalamic nuclei, hippocampus, orbitofrontal cortex (OFC), anterior cingulate gyrus (aCING), and peripeduncular nucleus of the brainstem, a network of regions implicated in the stress response. Arousal (EDA) correlated positively with brain activity in amygdala, OFC, and aCING during midcycle but not in early follicular, suggesting less cortical control of amygdala during early follicular, when arousal was increased. This is the first evidence suggesting that estrogen may likely attenuate arousal in women via cortical-subcortical control within HPA circuitry. Findings have important implications for normal sex-specific physiological functioning and may contribute to understanding higher rates of mood and anxiety disorders in women and differential sensitivity to trauma than men.

Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit.

Cocaine- and amphetamine-regulated transcript (CART) is present in a number of hypothalamic nuclei. Besides actions in circuits regulating feeding behaviour and stress responses, the hypothalamic functions of CART are largely unknown. We report that CART immunoreactivity is present in hypothalamic neuroendocrine neurones. Adult male rats received a systemic injection of the neuronal tracer Fluorogold (FG) 2 days before fixation, and subsequent double- and triple-labelling immunoflourescence analysis demonstrated that neuroendocrine CART-containing neurones were present in the anteroventral periventricular, supraoptic, paraventricular (PVN) and periventricular nuclei of the hypothalamus. In the PVN, CART-positive neuroendocrine neurones were found in all of cytoarchitectonically identified nuclei. In the periventricular nucleus, approximately one-third of somatostatin cells were also CART-immunoreactive. In the medial parvicellular subnucleus of the PVN, CART and FG coexisted with thyrotrophin-releasing hormone, whereas very few of the corticotrophin-releasing hormone containing cells were CART-immunoreactive. In the arcuate nucleus, CART was extensively colocalized with pro-opiomelanocortin in the ventrolateral part, but completely absent from neuroendocrine neurones of the dorsomedial part. To assess the possible role of CART as a hypothalamic-releasing factor, immunoreactive CART was measured in blood samples from the long portal vessels connecting the median eminence with the anterior pituitary gland. Adult male rats were anaesthetized and the infundibular stalk exposed via a transpharyngeal approach. The long portal vessels were transected and blood collected in 30-min periods (one prestimulatory and three poststimulatory periods). Compared to systemic venous plasma samples, baseline concentrations of immunoreactive CART were elevated in portal plasma. Exposure to sodium nitroprusside hypotension triggered a two-fold elevation of portal CART42-89 immunoreactivity throughout the 90-min stimulation period. In contrast, the concentration of portal plasma CART immunoreactivity dropped in the vehicle infused rats. The current study provides further evidence that CART is a neuroendocrine-releasing factor with a possible impact on anterior pituitary function during states of haemodynamic stress.

The hippocampus and depression.

The effect of depression on the hippocampus has become the focus of a number of structural and functional neuroimaging studies. In the past two decades, advances in neuroimaging techniques now allow the examination of subtle changes in both regional structure and function that are associated with the pathophysiology of depression. Many studies using 3-dimensional magnetic resonance imaging (MRI) volumetric measurement have reported decreases in hippocampal volume among depressed subjects compared with controls, whereas other studies have not found any volume loss. Differences among studies have been discussed. In some studies, the volume loss appears to have functional significance including an association with memory loss. Furthermore, we have found a trend towards loss of 5-HT(2A) receptors in the hippocampus using positron emission tomography (PET) to detect regional changes in [18F]altanserin binding. Functional imaging extends the sensitivity and specificity of structural imaging and will lead to a better understanding of affective disorders.

Hypothalamic accessory nuclei and their relation to the angiotensinergic and vasopressinergic systems.

The existence and colocalization of angiotensin II- and vasopressin-like immunoreactivity in individual magnocellular cell groups of the hypothalamus has been demonstrated by using immunocytochemical methods. These neurosecretory magnocellular groups consist of the paraventricular nucleus and the supraoptic nucleus, as well as different accessory cell groups. The fibers from the neurons of the accessory nuclei project directly to adjacent blood vessels and do not comigrate with the hypothalamo-neurohypophysial fiber pathway. On the basis of these findings it can be concluded that in the hypothalamus two different angiotensinergic and vasopressinergic neurosecretory systems exist: (1) an intrinsic hypothalamic and (2) a hypothalamo-neurohypophysial system. The distribution of the accessory cell groups in the hypothalamus is shown in a 3D reconstruction which includes the connection of these magnocellular nuclei with the vascular system in this area.

Effects of circulating tumor necrosis factor on the neuronal activity and expression of the genes encoding the tumor necrosis factor receptors (p55 and p75) in the rat brain: a view from the blood-brain barrier.

Tumor necrosis factor is a potent activator of myeloid cells, which acts via two cell-surface receptors, the p55 and p75 tumor necrosis factor receptors. The present study describes the cellular distribution of both receptor messenger RNAs across the rat brain under basal conditions and in response to systemic injection with the bacterial endotoxin lipopolysaccharide and recombinant rat tumor necrosis factor-alpha. Time-related induction of the messenger RNA encoding c-fos, cyclo-oxygenase-2 enzyme and the inhibitory factor kappa B alpha was assayed as an index of activated neurons and cells of the microvasculature by intravenous tumor necrosis factor-alpha challenge. The effect of the proinflammatory cytokine on the hypothalamic-pituitary-adrenal axis was determined by measuring the transcriptional activity of corticotropin-releasing factor and plasma corticosterone levels. Constitutive expression of p55 messenger RNA was detected in the circumventricular organs, choroid plexus, leptomeninges, the ependymal lining cells of the ventricular walls and along the blood vessels, whereas p75 transcript was barely detectable in the brain under basal conditions. Immunogenic insults caused up-regulation of both tumor necrosis factor receptors in barrier-associated structures, as well as over the blood vessels, an event that was associated with a robust activation of the microvasculature. Indeed, intravenous tumor necrosis factor-alpha provoked a rapid and transient transcription of inhibitory factor kappa B alpha and cyclo-oxygenase-2 within cells of the blood-brain barrier, and a dual-labeling technique provided the anatomical evidence that the endothelium of the brain capillaries expressed inhibitory factor kappa B alpha. Circulating tumor necrosis factor-alpha also rapidly stimulated c-fos expression in nuclei involved in the autonomic control, including the bed nucleus of the stria terminalis, the paraventricular nucleus of the hypothalamus, the central nucleus of the amygdala, the nucleus of the solitary tract and the ventrolateral medulla. A delayed c-fos mRNA induction was detected in the circumventricular organs, organum vascularis of the lamina terminalis, the subfornical organ, the median eminence and the area postrema. The paraventricular nucleus of the hypothalamus exhibited expression of corticotropin-releasing factor primary transcript that was associated with a sharp increase in the plasma corticosterone levels 1h after intravenous tumor necrosis factor-alpha administration. Taken together, these data provide the evidence that p55 is the most abundant tumor necrosis factor receptor in the central nervous system and is expressed in barrier-associated structures. Circulating tumor necrosis factor has the ability to directly activate the endothelium of the brain's large blood vessels and small capillaries, which may produce soluble molecules (such as prostaglandins) to vehicle the signal through parenchymal elements. The pattern of c-fos-inducible nuclei suggests complex neuronal circuits solicited by the cytokine to activate neuroendocrine corticotropin-releasing factor and the corticotroph axis, a key physiological response for the appropriate control of the systemic inflammatory response.

The inhibitory effect of hCG on counter current transfer of GnRH and the presence of LH/hCG receptors in the perihypophyseal cavernous sinus--carotid rete vascular complex of ewes.

The existence of the hormone passage from venous blood into arterial blood in the area of the perihypophyseal vascular complex has been demonstrated in some mammals, but its mechanism has not been defined. To study the regulatory mechanism we infused hCG into perihypophyseal cavernous sinus of ovariectomized, conscious ewes to test if the hCG would affect putative LH/hCG receptors and inhibit counter-current transfer of GnRH from the venous cavernous sinus to the arterial carotid rete. The latter study was done on an isolated head model. Ewes were ovariectomized in mid-anestrus and, after 4 to 5 wk were used in the experiments. On the day of experiment ewes were treated intramuscularly with estradiol benzoate or oil vehicle, and 18 to 20 h later were infused either with a multielectrolyte solution or hCG for 2 h via the venae angularis oculi. Immediately thereafter the ewes were anesthetized and exanguinated, and subsequently decapitated. The isolated head was perfused with Dextran in multielectrolyte. The 125I-GnRH was infused into the cavernous sinus via the venae angularis oculi for 5 min; contemporaneous samples were taken from the carotid rete and both jugular veins at 1-min intervals. Transfer of 125I-GnRH from the cavernous sinus to the carotid rete was inhibited by hCG in ewes pretreated with estradiol benzoate but not with oil (P<0.005). We collected tissue samples from the vascular complex of the cavernous sinus and carotid rete of cyclic ewes to determine the presence of LH/hCG receptors. In situ hybridization showed the presence of LH/hCG receptor mRNA transcripts in the walls of both arterial and venous compartments of the cavernous sinus-carotid rete complex, and immunohistochemistry revealed the presence of receptor proteins. These novel findings confirm previously obtained data suggesting that LH is a modulatory factor for the counter-current transfer of neuropeptides from the venous blood of the cavernous sinus to the arterial blood supplying the brain and hypophysis. The LH could modulate 125I-GnRH transfer acting directly on the vascular smooth muscle.

Regeneration of neurosecretory axons into various types of intrahypothalamic grafts is promoted by the absence of blood brain barrier: fine structural analysis.

Isogenous grafts of neural lobe and optic nerve and autologous grafts of sciatic nerve were placed into contact with the intrahypothalamically transected hypothalamo-neurohypophysial tract, and their fine structural characteristics examined at various time periods thereafter. The vascular bed of neural lobe grafts is composed primarily of fenestrated capillaries, that are permeable to blood-borne HRP throughout the entire experimental period. The microvasculature of sciatic nerve grafts consists of continuous, as well as fenestrated capillaries, which are similarly permeable to HRP. Fenestrated capillaries and HRP leakage in optic nerve grafts are observed at 10 days, but only in grafts located ventrally in the hypothalamus at 30 days. Neurosecretory axon regeneration is seen only in grafts or adjacent hypothalamus where the blood-brain barrier is breached. Regenerating axons are closely associated with the specific glial cells of the respective graft. Based on these observations, we conclude that blood-borne factors are necessary to initiate and sustain regeneration of transected neurosecretory axons, and that such regeneration occurs only in the presence of glial cells.

Adeno-pituitary hormones in human hypothalamic hypophysial blood.

An in vivo technique for collecting blood from the pituitary stalk using transphenoidal microsurgery has recently been developed in men with nonfunctioning pituitary disease. To determine the origin of this blood and the direction of the stream, we measured contemporaneously the levels of LH, FSH, PRL, GH, TSH, and ACTH in hypothalamic-hypophysial blood (HHB) and peripheral blood (PB). Eleven patients with nonfunctioning pituitary adenomas entered the study. The surgical procedure used for collecting HHB consisted of periodically aspirating small amounts of blood using a microsuction apparatus, just after tumor removal, kept in the postero-superior corner of the sella turcica at the junction of the diaphragm with the dursum sellae. The data show clearly the existence of a dramatic concentration gap in HHB vs. PB in all adeno-pituitary hormones (P = 0.003). The HHB/PB ratio varied from 50-600 in the different hormones. The secretion of adeno-pituitary hormones in blood drawn at the pituitary stalk level in man was reported for the first time. The dramatic HHB/PB ratio of the hormone levels has been emphasized. The most likely explanation for the markedly elevated hormone concentration gradient between central and peripheral blood was sampling of peri- and/or suprapituitary blood. To consider the origin and direction of the HHB stream, two hypotheses have been further advanced: 1) a retrograde bloodflow from the pituitary, and 2) a central-hypothalamic secretion.

Corticotrophin-releasing factor and arginine vasopressin in the hypothalamo-hypophyseal portal blood of rats following high-dose glucocorticoid treatment and withdrawal.

Hypothalamo-hypophyseal portal blood was obtained from rats treated with chronic, high-dose prednisolone in the drinking water and after subsequent withdrawal of the steroid for up to 7 days. Corticotrophin-releasing factor (CRF) immunoreactivity in portal blood was reduced by treatment with prednisolone but not completely abolished. There was a rapid recovery of CRF to control values within 3 days of withdrawal of prednisolone. There was no significant change in arginine vasopressin (AVP) in portal blood over the time-course of the experiment.

Antibodies in passive immunization studies: characteristics and consequences.

Antibodies to neuropeptides or hormones are frequently used in passive immunization studies to unravel their physiological role in signal transfer. In such in vivo experiments antibodies are considered to bind and thereby to biologically inactivate the endogenous substance during its journey from its site of secretion to its site of action (signalling time). However, little is known about the mechanism of action and characteristics of antibodies that determine such biological activity. Since the signalling time in neuronal and hormonal communication is short, the kinetics of antibody binding is an important feature. Here, we present a theoretical framework to describe antibody binding kinetics which can contribute to the design of passive immunization protocols. The specific effects of variation in antibody concentration, dissociation constant, and on-rate constant on these binding kinetics are demonstrated. Simple methods are described to determine these parameters, which may guide the selection of antibodies for passive immunization studies. When time is limited, the on-rate constant and the local antibody concentration are the most important determinants. Several points are illustrated for CRF signal transfer in the rat. CRF signalling time in the hypothalamo-pituitary complex, as established from dye transport experiments, was 3-7 sec. Based on parameters measured for a rat monoclonal antibody to CRF (PFU 83), we computed that half-maximal and full blockades of ether-induced ACTH secretion were associated with approximately 85% and more than 99% binding of CRF, respectively. From the theoretical framework presented in this study we conclude that, in general, the kinetics of antigen binding are sufficiently fast for antibodies to interfere with hormonal and probably nonsynaptic neuronal signal transfer. However, interference with fast signalling processes (less than 10 msec), which may occur in the brain, is unlikely.