The design of barriers in the hypothalamus allows the median eminence and the arcuate nucleus to enjoy private milieus: the former opens to the portal blood and the latter to the cerebrospinal fluid.

The blood-brain barrier (BBB) is a single uninterrupted barrier that in the brain capillaries is located at the endothelial cells and in the circumventricular organs, such as the choroid plexuses (CP) and median eminence (ME), is displaced to specialized ependymal cells. How do hypothalamic hormones reach the portal circulation without making the BBB leaky? The ME milieu is open to the portal vessels, while it is closed to the cerebrospinal fluid (CSF) and to the arcuate nucleus. The cell body and most of the axons of neurons projecting to the ME are localized in areas protected by the BBB, while the axon terminals are localized in the BBB-free area of the ME. This design implies a complex organization of the intercellular space of the median basal hypothalamus. The privacy of the ME milieu implies that those neurons projecting to this area would not be under the influence of compounds leaking from the portal capillaries, unless receptors for such compounds are located at the axon terminal. Amazingly, the arcuate nucleus also has its private milieu that is closed to all adjacent neural structures and open to the infundibular recess. The absence of multiciliated cells in this recess should result in a slow CSF flow at this level. This whole arrangement should facilitate the arrival of CSF signal to the arcuate nucleus. This review will show how peripheral hormones can reach hypothalamic targets without making the BBB leaky.

Neuroanatomical investigation of the gonadotrophin-releasing hormone 1 system in the seasonally breeding Cape dune mole-rat, Bathyergus suillus.

The gonadotrophin-releasing hormone 1 (GnRH1) system has been investigated immunohistochemically in Cape dune mole-rats (Bathyergus suillus), subterranean rodents that normally display severe aggression towards conspecifics. These animals breed seasonally and show a reduced mean plasma level of luteinising hormone during the non-breeding season. GnRH1-immunoreactive (ir) cell bodies and processes are found in the septal/preoptic area and the mediobasal hypothalamus; the cell bodies are found in equal measure in these two regions. Dense aggregations of GnRH1-ir fibres are present in the organum vasculosum of the lamina terminalis and the external zone of the median eminence. The total number of detectable GnRH1-ir cell bodies does not differ between the sexes or within the sexes between breeding and non-breeding seasons. Similarly there is no difference in the distribution of detectable GnRH1-ir cell bodies in male and female mole-rats in and out of the breeding season. Although the average size of GnRH1-ir cell bodies does not differ between the seasons in males, their size in females is significantly smaller in the non-breeding season. Whether this reduced size reflects reduced GnRH1 synthesis remains to be determined.

Distribution of somatostatin immunoreactive neurons and fibres in the central nervous system of a chondrostean, the Siberian sturgeon (Acipenser baeri).

Somatostatin (SOM) is a neuropeptide that is widely distributed in the central nervous system of vertebrates. Two isoforms of somatostatin (SS1 and SS2) have been characterized in sturgeon and in situ hybridisation studies in the sturgeon brain have demonstrated that mRNAs of the two somatostatin precursors (PSS1 and PSS2) are differentially expressed in neurons [Trabucchi, M., Tostivint, H., Lihrmann, I., Sollars, C., Vallarino, M., Dores, R.M., Vaudry, H., 2002. Polygenic expression of somatostatin in the sturgeon Acipenser transmontanus: molecular cloning and distribution of the mRNAs encoding two somatostatin precursors. J. Comp. Neurol. 443, 332-345.]. However, neither the morphology of somatostatinergic neurons nor the patterns of innervation have yet been characterized. To gain further insight into the evolution of this system in primitive bony fishes, we studied the distribution of somatostatin-immunoreactive (SOM-ir) cells and fibres in the brain of the Siberian sturgeon (Acipenser baeri). Most SOM-ir cells were found in the preoptic area and hypothalamus and abundant SOM-ir fibres coursed along the hypothalamic floor towards the median eminence, suggesting a hypophysiotrophic role for SOM in sturgeon. In addition, SOM-ir cells and fibres were observed in extrahypothalamic regions such as the telencephalon thalamus, rhombencephalon and spinal cord, which also suggests neuromodulatory and/or neurotransmitter functions for this peptide. Overall there was a good correlation between the distribution of SOM-ir neurons throughout the brain of A. baeri and that of PSS1 mRNA in Acipenser transmontanus. Comparative analysis of the results with those obtained in other groups of fishes and tetrapods indicates that widespread distribution of this peptide in the brain is shared by early vertebrate lines and that the general organization of the somatostatinergic systems has been well-conserved during evolution.

Role of androgens and glucocorticoids in the regulation of diazepam-binding inhibitor mRNA levels in male mouse hypothalamus.

In peripheral organs, gonadal and adrenal steroids regulate diazepam-binding inhibitor (DBI) mRNA expression. In order to further investigate the involvement of peripheral steroid hormones in the modulation of brain DBI mRNA expression, we studied by semiquantitative in situ hybridization the effect of adrenalectomy (ADX) and castration (CX) and short-term replacement therapy on DBI mRNA levels in the male mouse hypothalamus. Cells expressing DBI mRNA were mostly observed in the arcuate nucleus, the median eminence and the ependyma bordering the third ventricle. In the median eminence and the ependyma bordering the third ventricule, the DBI gene expression was decreased in ADX rats and a single injection of corticosterone to ADX rats induced a significant increase in DBI gene expression at 3 and 12 h time intervals without completely restoring the basal DBI mRNA expression observed in intact mice. In the arcuate nucleus, ADX and corticosterone administration did not modify DBI mRNA expression. CX down-regulated DBI gene expression in the ependyma bordering the third ventricle. The administration of dihydrotestosterone (3-24 h) completely reversed the inhibitory effect of CX. In the median eminence and arcuate nucleus, neither CX or dihydrotestosterone administration modified DBI mRNA levels. These results suggest that the effects of glucocorticoids on the hypothalamo-pituitary-adrenocortical axis and androgens on the hypothalamo-pituitary-gonadal axis are mediated by DBI.

Co-expression of vasopressin and androgen-binding protein in the rat hypothalamus.

In previous studies we have observed the expression of androgen binding protein (ABP) in the rat hypothalamo-neurohypophysial system. With immunocytochemical double staining we found partial co-localization with oxytocin. In the present study we used antibodies to the anti-diuretic hormone arginine vasopressin (AVP) for co-localization with ABP in the rat hypothalamus. Both antigens were seen in the magnocellular paraventricular and supraoptic nuclei. Dense fiber networks with varicosities containing both AVP and ABP immunoreactivity were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed also co-existence in the parvocellular portion of the paraventricular nucleus and in the suprachiasmatic nucleus. ABP immunoreactive neurons in the preoptic region were devoid of AVP staining, AVP neurons in the bed nucleus of the stria terminalis stained only occasionally for ABP. We conclude that both the magnocellular and the parvocellular hypothalamic vasopressin systems are capable of expressing the steroid binding globulin, which is probably subject to axonal transport, along with the peptide hormone. Intrahypothalamic expression of ABP may be among the mechanisms necessary for rapid actions of steroids on hypothalamic neuroendocrine systems.

A novel stereotaxic approach to the hypothalamus for the use of push-pull perfusion cannula in Holstein calves.

To determine secretory patterns of growth hormone-releasing hormone (GHRH) and somatostatin (SS) and their roles in the regulation of growth hormone (GH) secretion, a method for collecting hypothalamic perfusates, a push-pull perfusion method was developed in calves. With the use of the stereotaxic apparatus for cattle, a cannula was implanted into the hypothalamus of four male calves based upon cerebral ventriculography. Push-pull perfusates were collected at 10 min intervals for 6h and GHRH and SS concentrations in perfusates and plasma GH concentration were determined by EIAs and RIA, respectively. A cannula was implanted into the hypothalamus based on the image of the third ventricle and maintained for 1 month. GHRH and SS showed pulsatile secretion and the pulses for GHRH and SS were irregular in conscious animals. Neither GHRH nor SS secretion had a clear relationship with GH secretion. In the present study, we thus (1) established a stereotaxic technique for approaching the hypothalamus using cerebral ventriculography for calves, and (2) demonstrated that GHRH and SS secretion were pulsatile but not closely related to GH profile in conscious calves. The technique is useful for the study of the functions of the hypothalamus in the control of pituitary hormones in cattle.

Morphological aspects of the median eminence--place of accumulation and secretion of regulatory neurohormones and neuropeptides.

The median eminence (ME) is a small brain area forming both the structural and functional bridge between the hypothalamus and the hypophysis. It is supplied by a variety of neurohormones and neuropeptides which are delivered to the ME by different hypothalamic and extrahypothalamic pathways. These biologically active substances may act in the ME locally influencing the activity of secretion of the neighbouring terminals or, after being released from the neuronal endings into the network of fenestrated capillaries and transported to the hypophysis, they may be involved in the regulation of secretion of adenohypophyseal hormones. Recent demonstrations of extensive colocalizations of these biologically active substances in individual axonal endings in the ME with wide spectrum of biological actions further emphasizes the ME as an important place involved in the neuroendocrine regulatory processes.

Mapping of thyrotropin-releasing hormone (TRH) neuronal systems of rat forebrain projecting to the median eminence and the OVLT. Immunocytochemistry combined with retrograde labeling at the light and electron microscopic levels.

The thyrotropin-releasing hormone (TRH)-containing neurons that project to portal capillaries of the external zone of the median eminence (ME) and fenestrated capillaries of the organum vasculosum of the lamina terminalis (OVLT) were identified on thin paraffin and thick vibratome sections using a combination of retrograde labeling with peripherally administered Fluoro-Gold and fluorescence immunocytochemistry. The results indicate that the vast majority of those TRH neurons that project to the ME and the OVLT is located in the paraventricular nucleus (PVN), and most abundantly, in its medial parvicellular subdivision. Although numerous TRH-immunoreactive (TRH-i) neurons are present in other hypothalamic areas of the brain, only a few of them in the dorsal hypothalamic area behind the PVN and the periventricular preoptic nucleus could be retrogradely labeled. Since only a few Fluoro-Gold-accumulating and TRH-i perikarya were seen in other nuclei than the PVN, it is likely that the majority of nerve terminals in the OVLT also originates from TRH-i perikarya in the PVN. Fluoro-Gold, an electron-dense substance, is stored in the lysosomes of hypophysiotropic TRH-i perikarya and thus, it provides an excellent model for electron microscopic characterization of hypophysiotropic neurons at both the light and electron microscopic levels. The data together provides additional morphological evidence for the key role of the PVN in the regulation of TSH secretion.

Denervation of the rat posterior pituitary gland: validation of a stereotaxic method.

A stereotaxic surgical method was developed for interrupting the nerve fibres running through the rat pituitary stalk to the posterior pituitary gland without obliterating the hypothalamo-pituitary portal circulation. The pituitary stalk was compressed by the blunt tip of an L-shaped rotating knife. Successful operations produced mild diabetes insipidus, disappearance of arginine vasopressin from the neural lobe, accumulation of arginine vasopressin and neurosecretory material in the pituitary stalk and no infarction in the anterior lobe of the pituitary gland. In female rats, the oestrous cycle was only temporarily disturbed. Plasma prolactin and corticosterone levels were high during the first 24 h after the stalk compression but returned to normal baseline levels from the second day after the operation. One week after the operation plasma adrenocorticotropin and prolactin levels were in the control range while plasma alpha-melanocyte-stimulating hormone was elevated. Denervation of the posterior pituitary gland may help in studying the neural control of intermediate lobe function and the role of the neural lobe in various endocrine conditions, and may serve as a model for lesions of the pituitary stalk and formation of ectopic neurohypophysis in the human.

Corticotropin-releasing hormone neurons in the paraventricular nucleus project to the external zone of the median eminence: a study combining retrograde labeling with immunocytochemistry.

Corticotropin-releasing hormone (CRH) is the major regulator of the pituitary-adrenal axis. CRH-immunoreactive perikarya are widely distributed in the central nervous system; however, only those which participate directly in the regulation of adrenocorticotropin are connected to the portal circulation in the external zone of the median eminence. The present study describes the identification of these hypophysiotropic neurons using retrograde labeling and CRH immunocytochemistry. Fluoro-Gold was injected peripherally then, 5 days later, the animals were treated with colchicine. Twenty-four hours later the animals were sacrificed, and their brains were immunostained for CRH with the indirect immunofluorescence technique. The results indicate that the vast majority of the Fluoro-Gold-accumulating and CRH-immunopositive perikarya (hypophysiotropic neurons) are located in the medial parvicellular subdivision of the paraventricular nucleus (PVN). However, not each CRH-immunoreactive neuron contains Fluoro-Gold, i.e. a small portion of these neurons project to areas of the brain other than the median eminence. The anterior, lateral and periventricular subdivisions of the PVN also contain hypophysiotropic CRH-immunoreactive perikarya, however, their number is much less than in the medial parvicellular subdivision. Scattered double-labeled cells are also present in the medial preoptic area and the dorsal hypothalamus, just behind the PVN. These results support previous observations that the PVN, particularly the medial parvicellular subdivision, is the predominant source of the hypophysiotropic CRH neurons.

Neurokinin B peptide-2 neurons project from the hypothalamus to the thoracolumbar spinal cord of the rat.

The presence and location of CNS neurokinin B peptide-2-like immunoreactive neurons that project to the spinal cord were studied by a combination of retrograde transport of fluorescent dye (FluoroGold) and fluorescence immunocytochemistry. After injections of FluoroGold into the thoracic or lumbar segments of the rat spinal cord, serial sections of brain were stained with antisera directed against neurokinin B peptide-2. The results of the study showed that neurokinin B peptide-2-like immunoreactive neurons were located in the nucleus arcuate, median eminence, ventral and external bed nuclei of the stria terminalis, dorsal hypothalamic area, and medial habenula. Neurokinin B peptide-2 neurons that give rise to the long descending projections from the hypothalamus to thoracolumbar spinal cord were found only in the dorsal hypothalamic area. Approximately 36% of the neurokinin B peptide-2 neurons in the dorsal hypothalamic area projected to the spinal cord, whereas about 28% of the spinal projecting neurons in the dorsal hypothalamic area contained neurokinin B peptide-2-like immunoreactivity. Most of the spinal projecting neurokinin B peptide-2 neurons in the dorsal hypothalamic area had a cell size of 15 x 25 microns. In the spinal cord, immunoreactive neurokinin B peptide-2 fibers and terminals were distributed mainly in the superficial dorsal horn and the central autonomic area, with the highest density in laminae II and X, with less density in laminae IV and V. A few neurokinin peptide-2 fibers and terminals were also found in the ventral horn of the spinal cord. The results of the present study show that hypothalamic neurokinin B peptide-2 neurons are the main source of the spinal neurokinin B peptide-2.

Immunocytochemical localization of GnRH in the hypothalamus of the bat Miniopterus schreibersii schreibersii.

The distribution of gonadotropin-releasing hormone (GnRH) has been examined in the hypothalamus of adult female bats, Miniopterus schreibersii schreibersii, along annual reproductive cycle by means of light-microscopic immunocytochemistry. GnRH-immunoreactive cells are localized throughout the medial basal hypothalamus, being specially numerous in the arcuate nucleus. They are generally bipolar or monopolar neurons with smooth contour, although spiny cells were also found exclusively in the arcuate nucleus from periovulatory bats. Depletion in both number and immunocytochemical labelling of GnRH perikarya is detected in pregnant and lactating bats. GnRH-immunoreactive fibers are distributed in the internal layer of the median eminence and the infundibular stalk. Pregnant and lactating animals show lower GnRH fibers content and a less intense labelling than in periovulatory and hibernating conditions. The results suggest that changes in the secretory activity of GnRH neurons may be associated with hibernation and delayed implantation that suffer these mammalian species.

Sites of origin of growth hormone-releasing factor-containing neurons projecting to the stalk-median eminence of the rat.

The topographical location of neurons containing GRF which project to the median eminence were studied with immunofluorescence for GRF in combination with the retrograde transport of True blue. After the injection of True blue into the median eminence, retrogradely-labeled GRF neurons were identified in the arcuate nucleus and the lateral basal hypothalamus. GRF neurons in the perifornical area contained no positive dye. We concluded that the location of neurons containing hypophysiotrophic GRF are confined within the arcuate nucleus and the lateral basal hypothalamus.

CRF-containing neuron systems in the rat hypothalamus: retrograde tracing and immunohistochemical studies.

By employing a combination of the immunohistochemistry for rat corticotropin-releasing factor (rCRF) and retrograde tracing with biotinylated wheat germ agglutinin (b-WGA) injected into the posterior pituitary (group 1) or into the middle portion of the median eminence (group 2), functionally different populations of CRF neurons were identified in the rat hypothalamus. In the group 1 animals, WGA-labeling was seen not only in the posterior lobe but also in the intermediate lobe, in which CRF fibers exist. In these animals, WGA-labeling occurred for almost all large neurons in the anterior commissural, paraventricular (PV), and supraoptic (SO) nuclei, some of the neurons showing a slight immunoreactivity for anti-rCRF. Conversely, CRF positive neurons appeared in large numbers, some being labeled with WGA, in the caudal periventricular region (CPR), and in the dorsomedial to lateral hypothalamic area (DLH), especially in the latter. In the group 2 animals, WGA was disparsed throughout the subependymal, internal, and external layers of the medial portion of the median eminence, and was taken up by many small cells in the PV, almost half of the cells being immunoreactive for CRF. Slight WGA-labeling further occurred in some large neurons of the PV and SO, and in some cells of the DLH. It is concluded that hypophysiotropic CRF existing in the external layer of the median eminence originates from small neurons located in the PV, whereas CRF distributed in the posterior and intermediate pituitary originates from the magnocellular PV and SO, and from some neurons in the DLH and CPR.

Neuroendocrinology of pituitary hormone regulation.

Hormone secretion from the pituitary gland is dependent on the intimate association between the hypothalamus and pituitary by vascular and neuronal connections. The anatomic organization of these networks and their specific peptide and/or amide mediators are described in detail. Pertinent factors governing hypothalamic regulation of anterior pituitary secretion are also discussed.

A substance P-containing hypothalamic neuronal system projects to the median eminence.

Immunoreactive substance P (SPi) was measured in the various hypothalamic structures and amygdala one week after placing a cut around the medial basal hypothalamus (MBH). SPi content decreased in the stalk-median eminence (SME), anterior and posterior parts of the arcuate nucleus (inside the isolated region) as well as in the paraventricular nucleus (outside the cut), while there was no change in the posteromedial amygdaloid nucleus. We suggest that there is a tuberoinfundibular neuronal system containing SPi with cell bodies in the MBH and terminals in the SME. In addition, nerve fibers containing SPi from outside the MBH may reach both the SME and the arcuate nucleus and might influence neuroendocrine regulation.

Size, form and orientation of the neuronal perikarya and nuclei in the nucleus infundibularis of the male rat.

Size, form and orientation of nerve cells of the Nucleus infundibularis have been analyzed in frontal, horizontal and sagittal semithin sections of Vestopal-embedded hypothalami of the male rat. Morphometric studies were performed with a semiautomatic image analyzing system. By combining data from three different planes of section the three-dimensional size, shape and orientation of perikarya and nuclei in the different parts of the Nucleus infundibularis as well as gradients in mediolateral, rostrocaudal and ventrodorsal directions were examined. Additionally, cell density, volume and relation of the volumes of nucleus and perikaryon were determined. In all parts of the Nucleus infundibularis there occur cells exhibiting different sizes, forms and orientations. The mean values, however, show a rostrocaudal decrease and a mediolateral increase of the cell volumes with a maximum in the rostral, ventral and lateral parts and a minimum in the caudal, ventral and medial parts. In the entire Nucleus infundibularis there is a preferred orientation of the cell axes parallel to the wall of the third ventricle. Spherically-formed cells occur mainly in the rostral and basal parts, elongated and flattened cells in the caudal and dorsal parts. The nucleus-perikaryon relation is small in the rostral, lateral and ventral parts, large in the caudal, medial and dorsal parts. The cell density increases from rostral to caudal and decreases from medial to lateral. The results are discussed with respect to histochemical and immunohistochemical localization of substances relevant to neuroendocrine and neurotropic processes. This investigation exemplarily shows that different planes of section may lead to different descriptions of number and morphology of cells, and that a morphological analysis of nerve cells is only reliable from three-dimensional reconstruction using more than one direction of sectioning.

Light and electron microscopic immunocytochemistry of neurotensin-like immunoreactive neurons in the rat hypothalamus.

Neurotensin-like immunoreactive neuronal perikarya, fibers and terminals in the rat hypothalamus, particularly in the arcuate nucleus, the paraventricular nucleus and the median eminence, were investigated by light and electron microscopic immunocytochemistry. The main distributional areas of immunoreactive neuronal perikarya were found to be the arcuate nucleus, the periventricular nucleus and the paraventricular nucleus by light microscopic immunocytochemistry. Immunoreactive neuronal perikarya showed a characteristic distributional pattern in the arcuate nucleus. In the paraventricular nucleus they were distributed in both the magnocellular and parvocellular portions. A large number of immunoreactive terminals were observed throughout the external layer of the median eminence, particularly its lateral portion. A moderate number of immunoreactive terminals were also observed in the internal layer of the median eminence. By electron microscopic immunocytochemistry immunoreactive neuronal perikarya both in the arcuate and paraventricular nuclei showed generally well-developed cell organelles such as mitochondria, r-ER, and Golgi complex. In addition, immunoreactive dense granules were dispersed throughout the perikarya. A large number of immunoreactive terminals containing immunoreactive dense granules, clear vesicles and mitochondria were observed in the vicinity of pericapillary spaces of the external layer of the median eminence. This observation strongly suggests that neurotensin-like immunoreactive substance is released into the portal capillaries.

Distribution of immunoreactive substance P neurons in the hypothalamus and pituitary of the rhesus monkey.

The distribution of immunoreactive substance P (IR-SP) neurons was examined in the hypothalamus and pituitary gland of the rhesus monkey by using the peroxidase-antiperoxidase immunocytochemical technique. Immunoreactive SP cell bodies were observed in the arcuate nucleus, in the region lateral to the arcuate nucleus, and in the median eminence (ME). Immunoreactive SP cells were also seen in the periventricular area of the dorsal tuberal region. A rich network of SP fibers was concentrated in the arcuate region, and the fiber stain was particularly dense in the external zone of the median eminence and in the external layer of the infundibular stalk. Also, substance P fibers were seen in the internal layer of the pituitary stalk and in the neural lobe of the pituitary gland. Outside the hypothalamus a dense network of IR-SP fibers was observed in the globus pallidus.