Sim2 contributes to neuroendocrine hormone gene expression in the anterior hypothalamus

Paraventricular (PVN) and supraoptic nuclei of the hypothalamus maintain homeostasis by modulating pituitary hormonal output. PVN and supraoptic nuclei contain five major cell types: oxytocin-, vasopressin-, CRH-, somatostatin-, and TRH-secreting neurons. Sim1, Arnt2, and Otp genes are essential for terminal differentiation of these neurons. One of their common downstream genes, Brn2, is necessary for oxytocin, vasopressin, and CRH cell differentiation. Here we show that Sim2, a paralog of Sim1, contributes to the expression of Trh and Ss genes in the dorsal preoptic area, anterior-periventricular nucleus, and PVN. Sim2 expression overlaps with Trh- and Ss-expressing cells, and Sim2 mutants contain reduced numbers of Trh and Ss cells. Genetically, Sim1 acts upstream of Sim2 and partially compensates for the loss of Sim2. Comparative expression studies at the anterior hypothalamus at early stages reveal that there are separate pools of Trh cells with distinctive molecular codes defined by Sim1 and Sim2 expression. Together with previous reports, our results demonstrate that Sim1 and Otp utilize two common downstream genes, Brn2 and Sim2, to mediate distinctive sets of neuroendocrine hormone gene expression.     

DLK1 is a somato-dendritic protein expressed in hypothalamic arginine-vasopressin and oxytocin neurons

Delta-Like 1 Homolog, Dlk1, is a paternally imprinted gene encoding a transmembrane protein involved in the differentiation of several cell types. After birth, Dlk1 expression decreases substantially in all tissues except endocrine glands. Dlk1 deletion in mice results in pre-natal and post-natal growth deficiency, mild obesity, facial abnormalities, and abnormal skeletal development, suggesting involvement of Dlk1 in perinatal survival, normal growth and homeostasis of fat deposition. A neuroendocrine function has also been suggested for DLK1 but never characterised. To evaluate the neuroendocrine function of DLK1, we first characterised Dlk1 expression in mouse hypothalamus and then studied post-natal variations of the hypothalamic expression. Western Blot analysis of adult mouse hypothalamus protein extracts showed that Dlk1 was expressed almost exclusively as a soluble protein produced by cleavage of the extracellular domain. Immunohistochemistry showed neuronal DLK1 expression in the suprachiasmatic (SCN), supraoptic (SON), paraventricular (PVN), arcuate (ARC), dorsomedial (DMN) and lateral hypothalamic (LH) nuclei. DLK1 was expressed in the dendrites and perikarya of arginine-vasopressin neurons in PVN, SCN and SON and in oxytocin neurons in PVN and SON. These findings suggest a role for DLK1 in the post-natal development of hypothalamic functions, most notably those regulated by the arginine-vasopressin and oxytocin systems.     

BiP mRNA expression is upregulated by dehydration in vasopressin neurons in the hypothalamus in mice

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone that facilitates the proper folding of newly synthesized secretory and transmembrane proteins. Here we report that BiP mRNA was expressed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in wild-type mice under basal conditions. Dual in situ hybridization in the SON and PVN demonstrated that BiP mRNA was expressed in almost all the neurons of arginine vasopressin (AVP), an antidiuretic hormone. BiP mRNA expression levels were increased in proportion to AVP mRNA expression in the SON and PVN under dehydration. These data suggest that BiP is involved in the homeostasis of ER function in the AVP neurons in the SON and PVN.     

The Notch effector gene Hes1 regulates migration of hypothalamic neurons, neuropeptide content and axon targeting to the pituitary

Proper development of the hypothalamic-pituitary axis requires precise neuronal signaling to establish a network that regulates homeostasis. The developing hypothalamus and pituitary utilize similar signaling pathways for differentiation in embryonic development. The Notch signaling effector gene Hes1 is present in the developing hypothalamus and pituitary and is required for proper formation of the pituitary, which contains axons of arginine vasopressin (AVP) neurons from the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). We hypothesized that Hes1 is necessary for the generation, placement and projection of AVP neurons. We found that Hes1 null mice show no significant difference in cell proliferation or death in the developing diencephalon at embryonic day 10.5 (e10.5) or e11.5. By e16.5, AVP cell bodies are formed in the SON and PVN, but are abnormally placed, suggesting that Hes1 may be necessary for the migration of AVP neurons. GAD67 immunoreactivity is ectopically expressed in Hes1 null mice, which may contribute to cell body misplacement. Additionally, at e18.5 Hes1 null mice show continued misplacement of AVP cell bodies in the PVN and SON and additionally exhibit abnormal axonal projection. Using mass spectrometry to characterize peptide content, we found that Hes1 null pituitaries have aberrant somatostatin (SS) peptide, which correlates with abnormal SS cells in the pituitary and misplaced SS axon tracts at e18.5. Our results indicate that Notch signaling facilitates the migration and guidance of hypothalamic neurons, as well as neuropeptide content.     

TFF3 induced Fos protein expression in the magnocellular oxytocin neurons of the hypothalamus

TFF3 is synthesized in magnocellular oxytocin neurons of the supraoptic (SON) and paraventricular nuclei (PVN) of the rat and human hypothalamus. Here we investigated whether intracerebroventricular (i.c.v.) injection of TFF3 stimulates oxytocin release into the blood and activates Fos protein immunoreactivity in oxytocin neurons of the SON and PVN in rats. The results show that plasma oxytocin concentrations were not altered after i.c.v. injection of TFF3 or vehicle. Fos protein expression was significantly increased in both the SON and PVN after TFF3 injections and double labeling studies showed that the Fos signal was predominantly in oxytocin neurons.     

Reactions of oxytocin- and vasopressinergic cells of the hypothalamic supraoptic and paraventricular nuclei in the rat to repeated administrations of thyroliberin

Male Wistar rats were injected with 1 microgram thyroliberin twice a day for 8 days. Vasopressinergic (VE) and oxytocinergic (OE) cells of the supraoptic (SON) and paraventricular nuclei (PVN) were detected immunohistochemically, their changes were estimated morphometrically. The blood level of the thyrostimulating hormone determined by radioimmunoassay was 220% of the control on the 6th day but declined by the 9th day. The thyroid hormone concentration was also diminished. The nucleoli of the VE and OE cells of the SON were reduced by the 9th day (by 74 and 82%, respectively; P less than 0.01). The nucleoli of the OE cells of the PVN were enlarged to 120% (P less than 0.01), hence production of the neurohormone by these cells was intensified. The VE cells of the PVN remained essentially unchanged. The data obtained suggest that disagreement between the PVN and SON cell reactions is caused by various reactions of the nerve centers conveying toward the PVN and SON. Moreover, the OE and VE cells of the PVN apparently differ in their input. The importance of the OE cells of the PVN for the thyrostimulating hormone level normalization is suggested.     

Taurine reduces caspase-8 and caspase-9 expression induced by ischemia in the mouse hypothalamic nuclei

Summary. Taurine is a sulphur-containing amino acid abundant in the nervous system. It protects cells from ischemia-induced apoptosis, but the mechanism underlying this is not well established. The aim of our study was to explore the effects of taurine on two main pathways of apoptosis induced by ischemia: receptor-mediated and mitochondrial cell death. Brain slices containing the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus were incubated in vitro under control and simulated ischemic (oxygen-glucose deprivation for 30 min) conditions in the absence and presence of 20 mM taurine. Brain slices were harvested after the 180-min “postischemic” period and fixed in 4% paraformaldehyde. To estimate apoptosis, immunostaining was done for caspase-8 and caspase-9 in paraffin-embedded sections. Immunoreactive caspase-8 and caspase-9 cells were observed in SON and PVN in all experimental groups, but in the “ischemic” group the expression of caspase-8 and caspase-9 and the number of immunoreactive cells was significantly increased in both hypothalamic nuclei. Addition of taurine (20 mM) to the incubation medium induced a marked decrease in caspase-8 and caspase-9 immunoreactivity after ischemia in SON and PVN when compared with the taurine-untreated “ischemic” group. Taurine reduces ischemia-induced caspase-8 and caspase-9 expression, the key inductors of apoptosis in SON and PVN. Authors’ address: Dr. Andrey Taranukhin, Tampere Brain Research Center, Medical School, University of Tampere, FI-33014 Finland     

Rhythmicity of the activity of the supraoptic and paraventricular nuclei of the hypothalamus of the C57Bl mouse

Rhythmical changes in the activity of the neurosecretory processes have been compared in the supraoptic (SON) and paraventricular (PVN) nuclei in the C57Bl mice hypothalamus during vernal equinox. Parts of the neurosecretory cells, being at stages of synthesis, excretion and accumulation of secrete, volumes of the cell nuclei and nucleoli survey as criteria of their activity. Similar feature for the rhythmic of both nuclei studied is the highest activation of the processes during day time, when mice are resting; this is demonstrated as the maximal amount of actively synthesizing cells, maximal volumes of the cell nuclei and nucleoli. The peculiarities of the rhythmic display in the activity is manifested as a greater ability of the SON cells to accumulate neurosecrete. The accumulation of the secretory material in the SON cells precedes to the period of its maximal activity (1-7 PM) characterized: by making the cells free from the secrete and by a maximal increasing the volume of the nucleoli. In the PVN intensified display of the activity is noted at early hours of the day, and the amount of the cells not containing the secrete--at 6 PM. Lack of the neurosecrete accumulation in the PVN cells speaks in favour of more steady than in the SON cells excretion of the secrete. This demonstrates a more even maintenance of neurohormones concentration in the organism.     

Regulation of Apoptosis in Nonapeptidergic Neurons of Rat Hypothalamus by Catecholamines in Experiments in vitro

Effects of noradrenaline (NA) and dopamine (DA) on apoptosis of nonapeptidergic neurons of supraoptic (SON) and paraventricular (PVN) nuclei of hypothalamus of male Wistar rats was studied in experiments in vitro. Incubation of hypothalamic sections in the medium with added NA was shown to induce an increase of the amount of pro-apoptotic protein caspase-9 in the nonapeptidergic neurons of the SON and PVN. A comparison of the level of neuronal NO-synthase with the level of caspase-9 expression in these neurons allows concluding that NA leads to initiation of apoptosis in neurons of the SON with mediation by nitric oxide (NO). In the PVN, the NA-induced initiation of apoptosis does not depend on the NO level. Addition of DA to the incubation medium results in an increase of the caspase-9 amount only in PVN neurons regardless of the NO content. The absence of neuronal death after the NA-induced increase of the caspase-9 level in the cells of SON and PVN seems to be due to increased expression of the anti-apoptotic protein bcl-2. Protection of the PVN neurons from death after addition of DA to the incubation medium is probably independent of the expression level of bcl-2. Thus, in the nonapeptidergic neurons of the SON and PVN, which are related by origin and by performed functions, modulation of the process of apoptosis by elevated concentrations of NA and DA is realized by different mechanisms.     

Neural Connections Between Prosencephalic Structures Involved in Vasopressin Release

1. The diagonal band (DB) and the lateral septal area (LSA) are two prosencephalic structures, which were implicated in vasopressin release.2. The present experiment was designed to investigate neural connections between the DB and the LSA and from these nuclei to the paraventricular (PVN) and supraoptic (SON) nuclei, which could be related to vasopressin release.3. For the above purpose the bidirectional neuronal tracer biotinylated dextran amine (BDA) was injected into the DB or the LSA of male Wistar rats. Five days later the animals were sacrificed and brain slices were processed and analyzed to determine neuronal projections efferent from as well as afferent to these structures.4. Neuronal staining was more prominent in regions ipsilateral to the BDA injection site.5. After BDA injections into the DB, efferent projections from the DB were observed at the LSA, the PVN, the prefrontal cortex, the mediodorsal thalamic nucleus, and throughout the anterior hypothalamus, but not at the SON. At the PVN, labeled varicose fibers were observed at the magnocellular portion. The DB was found to receive a massive input from the LSA. More discrete projections to the DB were originated at the prefrontal cortex and from hypothalamic neurons outside the PVN and the SON.6. After BDA injections into the ventral portion of the LSA, efferent projections from the LSA were intense at the DB and throughout the hypothalamus. Labeled fibers were observed at the structures surrounding the SON or the PVN but not within those nuclei.7. The results indicate a massive neural output from the LSA to the DB and the existence of a direct neural connection from the DB to the PVN. No direct connections were observed between the LSA and the magnocellular nuclei (PVN and SON) or between the DB and the SON.     

Maturation of the human hypothalamo-hypophyseal neurosecretory system in prenatal ontogeny. A light-optical and electron microscopic study

The hypothalamic-hypophysial neurosecretory system (HHNS) was studied in the human foetuses from the age of 8 weeks till birth. The hypothalamus of 8 weeks old foetuses is weakly differentiated, no individual cell groups, so-called nuclei, are identified. The supraoptic (SON) and paraventricular (PVN) nuclei are identified from the age of 12 weeks on. The size of cell nuclei increases with the age. The Homori-positive granules were first found in some SON and PVN cell and in neurohypophysis in the 18 weeks old foetuses. It was shown under the electron microscope that the neurohypophysis of 8 weeks old foetuses consisted mainly of pituicytes with axons among the cytoplasmic processes of the latter. After the age of 10 weeks, the area of parenchyma of the neurohypophysis occupied by axons increased and typical elementary neurosecretory granules appeared in them. The data obtained are discussed with respect to the participation of HHNS in the regulation of water metabolism in the human foetuses.     

Genomic Effects of Cold and Isolation Stress on Magnocellular Vasopressin mRNA-Containing Cells in the Hypothalamus of the Rat

We assessed the effects of cold and isolation stress on arginine vasopressin (AVP) mRNA in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Vasopressin mRNA levels were determined by in situ hybridization histochemistry at the cellular level. In posterior magnocellular neurons of the PVN isolation stress for 7 or 14 days increased vasopressin mRNA levels 28 and 29%, respectively, compared to group-housed controls. No significant alterations in vasopressin gene expression were observed in the SON after 7 or 14 days of isolation stress. Scattered magnocellular AVP mRNA-expressing cells of the medial parvocellular PVN showed increases of 19 and 34% after 7 and 14 days of isolation, respectively. We also studied the effect of cold or combined cold and isolation stress on vasopressin gene expression in the PVN and SON. Cold stress for 3 h daily for 4 consecutive days increased AVP mRNA levels in the posterior magnocellular PVN by 15%. Cold-isolated animals showed an increase of 21%. No significant effect on AVP mRNA levels in the SON was observed. In contrast to the posterior magnocellular PVN, cold or cold-isolation stress increased AVP mRNA in magnocellular neurons of the medial parvocellular region of the PVN by 25 and 43%, respectively, relative to control rats. These results suggest that psychological and metabolic stress may be added to the list of stressors that activate the hypothalamo-neurohypophysial system.     

Immunohistochemical expression of Bcl-2, p53 and caspase-9 in hypothalamus magnocellular centers of nNOS knockout mice following water deprivation

Abstract

We studied the interactions between apoptosis regulator proteins (Bcl-2, p53 and caspase-9) and neuronal nitric oxide in vasopressinergic magnocellular centers of the hypothalamus using neuronal nitric oxide synthase (nNOS) gene knockout mice. nNOS gene deletion resulted in accumulation of Bcl-2, p53 and caspase-9 in the paraventricular (PVN) and supraoptic (SON) nuclei in controls. Dehydration increased the levels of all three apoptosis regulator proteins studied in nuclei of wild type mice. In the hypothalamus magnocellular centers of nNOS knockout mice, however, expression of Bcl-2, p53 and caspase-9 was unchanged after dehydration. The number of magnocellular neurons did not change in the SON and PVN of nNOS deficient mice compared to wild type, and after dehydration, cell death was not observed in either nucleus of wild type or knockout mice despite activation of apoptosis regulator protein expression. Thus, we demonstrated that gene disruption of nNOS prevents activation of Bcl-2, p53 and caspase-9 expression during water deprivation, and that nNOS deficiency did not affect survival of magnocellular neurons of the hypothalamus.     

Immunohistochemical investigation of Bcl-2 and p53 levels in rat hypothalamus after sleep deprivation

On Wistar rats in view of electrophysiological parameters after sleep deprivation (SD; awake by gentle handling method) and the subsequent postdeprivative sleep (PDS) immunohistochemical investigation of Bcl-2 and p53 peptides optical density levels in neurons of paraventricular (PVN), supraoptic (SON) and median (MnPN) hypothalamus nuclei was carried out. The Bcl-2 was increased in all nuclei both after SD and PDS. The level of p53 was increased in PVN and SON after SD and PDS, but in MnPN only on PDS. Any morphological attributes of apoptosis in the nuclei was not revealed. Obtained data testify an active role of p53 and Bcl-2 peptides in regulation of neuronal activity in hypothalamus at change of a cycle wakefulness-sleep.     

Microarray analysis reveals interleukin-6 as a novel secretory product of the hypothalamo-neurohypophyseal system

Physiological activation of the hypothalamo-neurohypophyseal system (HNS) by dehydration results is a massive release of vasopressin (VP) from the posterior pituitary. This is accompanied by a functional remodeling of the HNS. In this study we used cDNA arrays in an attempt to identify genes that exhibit differential expression in the hypothalamus following dehydration. Our study revealed nine candidate genes, including interleukin-6 (IL-6) as a putative novel secretory product of HNS worthy of further analysis. In situ hybridization and immunocytochemistry confirmed that IL-6 is robustly expressed in the supraoptic (SON) and the paraventricular (PVN) nuclei of the hypothalamus. By double staining immunofluorescence we showed that IL-6 is largely co-localized with VP in the SON and PVN. In situ hybridization, immunocytochemistry, and Western blotting all revealed IL-6 up-regulation in the SON and PVN following dehydration, thus validating the array data. The same dehydration stimulus resulted in an increase in IL-6 immunoreactivity in the axons of the internal zone of the median eminence and a marked reduction in IL-6-like material in the posterior pituitary gland. We thus suggest that IL-6 takes the same secretory pathway as VP and is secreted from the posterior pituitary following a physiological stimulus.     

Functional difference between the magnocellular neurosecretory nuclei of the rat hypothalamus

The supraoptic, paraventricular, and postoptic nuclei (SON, PVN, and PON, respectively) of the hypothalamus were studied under conditions of 3 months training of rats to hypoxia (exposure for 6 h daily in a low pressure chamber under 7600m of simulated altitude). All the three nuclei were activated during the first 5 days, and then the state of the SON cells normalized. Functional activity of the PVN and PON decreased (the nucleolar volume of the neurosecretory cells diminished to 70--80%, the amount of the neurosecretory substance in the cells and the posterior lobe of the hypophysis was reduced). Such a decreased activity of the PVN and PON persisted till the end of the experiment. A positive correlation of the thyroid epithelium height and the nucleolar volume of the PVN and PON cells was established for both the PVN (r=0.81, P less than 0.05) and the PON (r=0.82; P less than 0.05); no significant correlation was revealed for the SON (r=0.51; P less than 0.05). Thus, functional similarity of the PVN and the PON, and some peculiarities in the SON reaction under conditions of the experiment described was demonstrated.