A molecular census of arcuate hypothalamus and median eminence cell types.

The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a new leptin-sensing neuron population, multiple agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) subtypes, and an orexigenic somatostatin neuron population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinct responses in AgRP and POMC neuron subtypes. Finally, integrating our data with human genome-wide association study data implicates two previously unknown neuron populations in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred.

A mammalian neural tissue opsin (Opsin 5) is a deep brain photoreceptor in birds.

It has been known for many decades that nonmammalian vertebrates detect light by deep brain photoreceptors that lie outside the retina and pineal organ to regulate seasonal cycle of reproduction. However, the identity of these photoreceptors has so far remained unclear. Here we report that Opsin 5 is a deep brain photoreceptive molecule in the quail brain. Expression analysis of members of the opsin superfamily identified as Opsin 5 (OPN5; also known as Gpr136, Neuropsin, PGR12, and TMEM13) mRNA in the paraventricular organ (PVO), an area long believed to be capable of phototransduction. Immunohistochemistry identified Opsin 5 in neurons that contact the cerebrospinal fluid in the PVO, as well as fibers extending to the external zone of the median eminence adjacent to the pars tuberalis of the pituitary gland, which translates photoperiodic information into neuroendocrine responses. Heterologous expression of Opsin 5 in Xenopus oocytes resulted in light-dependent activation of membrane currents, the action spectrum of which showed peak sensitivity (lambda(max)) at approximately 420 nm. We also found that short-wavelength light, i.e., between UV-B and blue light, induced photoperiodic responses in eye-patched, pinealectomized quail. Thus, Opsin 5 appears to be one of the deep brain photoreceptive molecules that regulates seasonal reproduction in birds.

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.

Sexual dimorphism in the organization of the rat hypothalamic infundibular area.

The hypothalamic infundibular area is located outside the blood-brain barrier and includes, the ventromedial arcuate nucleus (vmARC) sensing circulating substances, and the median eminence (ME) where neurohormones are released into the hypothalamo-hypophysial vasculature. This integrated functional unit, pivotal in endocrine control, adjusts neuroendocrine output to feedback information. Despite a differing physiology in males and females, this functional unit has not appeared differently organized between sexes. Using immunocytochemistry, we describe here for the first time in adult rats, a conspicuous sex-difference in its axonal wiring by intrinsic glutamatergic neurons containing the neuropeptides neurokinin B (NKB) and dynorphin. In the male, NKB neurons send axons to capillary vessels of the vmARC and of the ME (only where gonadotropin-releasing hormone (GnRH) axons terminate). Electron microscopy revealed that NKB axons target the barrier of tanycytes around fenestrated capillary vessels (in addition to GnRH axons), suggesting a control of regional bidirectional permeability. In the female, NKB neurons send axons to the neuropile of the vmARC, suggesting a direct control of its sensor neurons. The other projections of NKB neurons, studied by surgical isolation of the ARC-ME complex and confocal microscopy, are not sexually dimorphic and target both integrative and neuroendocrine centers controlling reproduction and metabolism, suggesting a broad influence over endocrine function. These observations demonstrate that the mechanisms subserving hypothalamic permeability and sensitivity to feedback information are sexually dimorphic, making the infundibular area a privileged site of generation of the male-to-female differences in the adult pattern of pulsatile hormonal secretions.

Brain-derived neurotrophic factor in the brain of Xenopus laevis may act as a pituitary neurohormone together with mesotocin.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, occurs abundantly in the brain, where it exerts a variety of neural functions. We previously demonstrated that BDNF also exists in the endocrine melanotroph cells in the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis, suggesting that BDNF, in addition to its neural actions within the brain, can act as a hormone. In the present study, we tested whether BDNF, in addition to its neural and hormonal roles, can be released as a neurohormone from the neural pituitary lobe of X. laevis. By light immunocytochemistry, we show that BDNF is present in perikarya, in ventrolaterally projecting axons of the hypothalamic magnocellular nucleus and in the neural lobe of the pituitary gland, and that it coexists in these structures with the amphibian neurohormone, mesotocin. The neural lobe was studied in detail at the ultrastructural level. Two types of neurohaemal axon terminals were observed, occurring intermingled and in similar numbers. Type A is filled with round, moderately electron-dense secretory granules with a mean diameter of approximately 145 nm. Type B terminals contain electron-dense and smaller, ellipsoid granules (long and short diameter approximately 140 and 100 nm, respectively). BDNF is exclusively present in secretory granules of type A axon terminals. Double gold-immunolabelling revealed that BDNF coexists in these granules with mesotocin. Furthermore, we demonstrate in an superfusion study performed in vitro that mesotocin stimulates peptide release from the endocrine melanotroph cells. On the basis of these data, we propose that BDNF can act on these cells as a neurohormone.

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.

Histological and histochemical study of the magnocellular neuroendocrine system in white rat after parenteral administration of adrenaline.

The effect of a single dose of adrenaline on the neurosecretory action of the hypothalamo-hypophyseal system was investigated in 90 male rats. Increased release and transport of neurosecretory material were observed after a low dose of adrenaline, and the enhancement of the neurosecretory process was noted following a high dose. As shown in numerous reports, there is a link between the neuroendocrine system and the hypothalamic adrenergic system.

Transient structures of the human fetal brain: subplate, thalamic reticular complex, ganglionic eminence.

Morphological features of the subplate, the thalamic reticular complex and the ganglionic eminence, which represent three major transient structures of the human fetal forebrain, are summarized with special reference to their functional roles. The subplate harboring various neuronal types is an outstandingly wide zone subjacent to the cortical plate in the human fetal brain. Within the subplate various cortical afferents establish synaptic contacts for a prolonged period before entering the cortical plate. Therefore, the subplate is regarded as a "waiting compartment" which is required for the formation of mature cortical connections. Next to the thalamic reticular nucleus, within the fibers of internal capsule, the perireticular nucleus is located which has been established as a distinct entity during development. Its various neuronal types express a number of different neuroactive substances. Perinatally, the perireticular nucleus is drastically reduced in size. It is involved in the guidance of corticofugal and thalamocortical fibers. The ganglionic eminence is a conspicuous proliferative area that persists throughout nearly the entire fetal period. In the human fetal brain it extends medially upon the dorsal thalamic nuclei which receive precursor cells from the ganglionic eminence. Postmitotic cells in the marginal zone of the ganglionic eminence serve as an intermediate target for growing axons. On the whole, all three structures establish transient neural circuitries that may be essential for the formation of adult projections. The characteristics of the three transient structures are particularly relevant for developmental neuropathology as these structures may be damaged in disorders that preferentially occur in preterm infants.

Detailed magnetic resonance imaging anatomy of the cisternal segment of the abducent nerve: Dorello's canal and neurovascular relationships and landmarks.

OBJECT: The goal of this study was to identify reliably the cisternal segment of the abducent nerve by using the three-dimensional Fourier transform constructive interference in steady-state (3-D CISS) magnetic resonance (MR) imaging sequence to define landmarks that assist in the identification of the abducent nerve on MR imaging and to describe the nerve's relationship to the anterior inferior cerebellar artery (AICA). METHODS: A total of 26 volunteers underwent 3-D CISS MR imaging, and 10 of these volunteers also underwent MR angiography in which a time-of-flight sequence was used to identify the facial colliculus, the abducent nerve and its apparent origin, Dorello's canal, and the AICA. The authors identified the abducent nerve with certainty in 96% of 3-D CISS sequences obtained in the axial and sagittal planes and in 94% obtained in the coronal plane. The nerve emerged from the pontomedullary sulcus in 94% of cases. The facial colliculus could always be identified, and Dorello's canal was identified in 94% of cases. In 76.6% of cases, the abducent nerve was seen to contact the AICA, which passed inferior to the nerve in 63.8% of cases and superior to it in 29.8%. CONCLUSIONS: The anatomical course of the abducent nerve and its relationship to the AICA and other blood vessels can be reliably identified using a 3-D CISS MR sequence with the facial colliculus and Dorello's canal serving as landmarks.

Median eminence lesions reveal separate hypothalamic control of pulsatile follicle-stimulating hormone and luteinizing hormone release.

The effects of hypothalamic lesions designed to destroy either the anterior median eminence (ME) or the posterior and mid-ME on pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined in castrated male rats. In sham-operated animals, mean plasma FSH concentrations rose to peak at 10 min after the onset of sampling, whereas LH declined to a nadir during this time. In the final sample at 120 min, the mean FSH concentrations peaked as LH decreased to its minimal value. In rats with anterior ME lesions, there was suppression of LH pulses with continuing FSH pulses in 12 of 21 rats. On the other hand, in animals with posterior to mid-ME lesions, 3 out of 21 rats had elimination of FSH pulses, whereas LH pulses were maintained. Fifteen of 42 operated rats had complete ME lesions, and pulses of both hormones were abolished. The remaining 12 rats had partial ME lesions that produced a partial block of the release of both hormones. The results support the concept of separate hypothalamic control of FSH and LH release with the axons of the putative FSH-releasing factor (FSHRF) neuronal system terminating primarily in the mid- to caudal ME, whereas those of the LHRH neuronal system terminate in the anterior and mid-median eminence. We hypothesize that pulses of FSH alone are mediated by release of the FSHRF into the hypophyseal portal vessels, whereas those of LH alone are mediated by LHRH. Pulses of both gonadotropins simultaneously may be mediated by pulses of both releasing hormones simultaneously. Alternatively, relatively large pulses of LHRH alone may account for simultaneous pulses of both gonadotropins since LHRH has intrinsic FSH-releasing activity.

Somatostatin (SRIF) like immunoreactivity in median eminence (ME) of female rat brain: evidence of compartmentalization in ME.

Somatostatin (SRIF) immunoreactivity was observed in rostrocaudal extent (Bregma levels-1.8 mm to -3.8 mm) of the median eminence (ME) in female rat brain using Avidin-Biotin Complex (ABC) method (Hsu et al, 1981). SRIF immunoreactivity (IR) was observed in entire-rostrocaudal extent of both internal (IZ) and external zone (EZ) of ME. Image analysis of SRIF stained sections showed that in rostral ME (Bregma -1.8 to -2.3 mm) dense immunoreactive nerve terminals were observed in EZ. In medial ME (Bregma -2.3 mm to 3.3 mm) SRIF-IR was low in IZ and dense in EZ. In this region dense immunoreactive nerve terminals were observed in lateral margin of EZ. In caudal ME (Bregma -3.3 mm to -3.8 mm) nerve terminals in lateral EZ and median IZ and EZ showed dense reactivity in nerve terminals. These results led us to hypothesize that each region-lateral IZ and EZ and medial IZ and EZ are independent functional units in ME. Six functionally independent compartments could be identified-Compartment I and III of IZ and IV in EZ (Lateral margins in ME), Compartment V (IZ) and Compartment VI (medial EZ).

Factors influencing maternal behavior in the hubb/hubb mutant mouse.

We examined the maternal behavior of hubb/hubb mutant mice and normal control (+/hubb) siblings. From previous observations we noted that mutants groom their pups less, suckle less than normal, and often cannibalize the young. To date, these observations had not been quantified. Although prolactin (PRL) is linked to maternal behavior, it was difficult to measure because of the hyperirratibility of the mutant mice. Consequently, dopamine (DA) and its metabolite, dihydroxyphenylacetic acid (DOPAC), were measured in the median eminence in brains of both normal and mutant mice. Tyrosine hydroxylase, the rate-determining step in dopamine synthesis, was localized in the brain by immunohistochemistry. Five mutant and nine normal dams were observed for pup retrieval and crouching. Mean time for pup retrieval was slower (p < 0.06) for mutants (28.09 s) than for normal dams (18.49 s). Crouching was the same for both strains. Mutant pups were cold to the touch, and not well groomed. Brains from both strains were examined at Day 11 and Day 18 of gestation and Day 2 and Day 11 of lactation. Qualitatively, tyrosine hydroxylase localization in the arcuate nucleus and median eminence was the same in both strains for the gestation samples. The decrease in staining observed from gestation to lactation in the normal mice was increased in the mutants. Dopamine was similar in both strains at all stages, but DOPAC was significantly higher at early lactation in the mutants. We do not assume an absolute inverse relationship between dopaminergic activities and prolactin, but it is likely that the increase in DOPAC in the mutant reflects a decrease in prolactin, which could contribute to the diminished maternal care in the mutants.

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.