Sagittalis nucleus: a novel hypothalamic nucleus.

The sagittalis nucleus (SGN) of the hypothalamus is a newly-identified nucleus that is located in the interstitial area between the arcuate and ventromedial nuclei of the rat hypothalamus and for which the long axis of the nucleus is oriented sagittally. Interestingly, the SGN exhibits structural and physiological sex differences, as defined by Nissl staining and oestrogen receptor (ER)alpha immunoreactivity (-ir), being larger in males than females. The structural sex difference is established by sex steroid action in neonates because the treatment of female pups with testosterone propionate masculinised the SGN. The phenotypical sex difference in ERalpha-ir is mediated hormonally in adulthood. Ovariectomy of female rats caused a significant increase in ERalpha-ir in the SGN, and eliminated the physiological sex difference, but with recovery to the level of gonad-intact females when given oestradiol replacement. Adult females have oestrous cycle-related variations in ERalpha-ir in the SGN, with levels at a nadir during the evening of pro-oestrous. The discovery of the SGN, a target of sex steroid action, provides a new opportunity for explaining hormonal regulation of sexually-differentiated behavioural and endocrine functions.

Physiological studies of stress responses in the hypothalamus of vasopressin-enhanced green fluorescent protein transgenic rat.

Arginine vasopressin (AVP) plays an important role in stress-induced activation of the hypothalamic-pituitary adrenal axis. In the present study, AVP-enhanced green fluorescent protein (eGFP) transgenic rats were used to investigate changes in AVP-eGFP expression in the hypothalamic paraventricular nucleus (PVN) and the median eminence (ME) upon exposure to stress conditions. The eGFP fluorescence in the parvocellular division of the PVN (pPVN) was markedly increased 5 days after bilateral adrenalectomy (ADX) and it was colocalised with corticotrophin-releasing hormone-like immunoreactivity in the pPVN. Peripheral administration of dexamethasone completely suppressed the increase of eGFP fluorescence in the pPVN and the external layer of the ME (eME) after bilateral ADX. Significant increases of eGFP fluorescence were observed in the pPVN 6, 12, 24 and 48 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS). In the eME, eGFP fluorescence was significantly increased 48 h after i.p. administration of LPS. By contrast, eGFP fluorescence changed neither in the magnocellular division of the PVN, nor the internal layer of the ME after i.p. administration of LPS. Our results indicate that AVP-eGFP transgenic rats are useful animal model to study dynamic changes of AVP expression in the hypothalamus under stressful conditions.

Motoneuron-specific expression of NR3B, a novel NMDA-type glutamate receptor subunit that works in a dominant-negative manner.

We have identified a novel glutamate receptor subunit on the human and mouse genome. Cloning of the mouse cDNA revealed a protein consisting of 1003 amino acids encoded by at least nine exons. This protein showed the highest similarity (51%) to the NR3A subunit of the NMDA receptor and therefore was termed NR3B. NR3B has a structure typical of glutamate receptor family members with a signal peptide and four membrane-associated regions. Amino acids forming a ligand-binding pocket are conserved. When coexpressed with NR1 and NR2A in heterologous cells, NR3B suppressed glutamate-induced current similarly to NR3A. Thus members of the NR3 class of NMDA receptors act as dominant-negative subunits in the NMDA receptor complex. NR3B shows very restricted expression in somatic motoneurons of the brainstem and spinal cord. Its expression in other types of motoneurons, including autonomic motoneurons in Onuf's nucleus and oculomotor neurons, is significantly weaker. Our results indicate that NR3B is important as a regulatory subunit that controls NMDA receptor transmission in motoneurons. It may be involved in the pathogenesis of neurodegenerative diseases involving motoneurons as well.

Cloning and characterization of LUN, a novel ring finger protein that is highly expressed in lung and specifically binds to a palindromic sequence.

We isolated cDNAs encoding a novel RING finger protein (LUN), the mRNAs of which were expressed at high levels in the lung. In situ hybridization revealed that LUN mRNAs were expressed in the alveolar epithelium of the lung. The LUN gene locus was assigned to chromosome 9p21, which contains candidate tumor suppressor genes associated with loss of heterozygosity in more than 86% of small cell lung cancers. We clarified that LUN is localized to the nucleus and reveals Zn(2+)-dependent DNA binding activity. The region from amino acids 51 to 374 of LUN is responsible for DNA binding. Furthermore, we identified a novel palindromic binding consensus (5'-TCCCAGCACTTTGGGA-3') for the LUN binding. Interestingly, this LUN binding palindromic sequence is found in the upstream transcriptional regulatory region of the E-cadherin gene and two intervening regions of the talin gene. Our results suggested that LUN might be an important trans-acting transcriptional regulator for lung cancer-associated genes including E-cadherin and talin genes.

Immunohistochemical study of nucleoporin p62 in the hippocampus and hypothalamus of the rat brain.

We immunohistochemically studied the distribution of nucleoporin p62 in the hippocampus and hypothalamus of rat brain. Previous reports have shown the presence of p62-immunoreactivity (ir) in the nuclear rim in the non-neuronal cells, but the present study showed that of p62-ir within the nucleus in addition to the nuclear rim in the neuronal cells of the hippocampus and hypothalamic nuclei; in these areas the glucocorticoid receptor (GR) undergoes nucleocytoplasmic translocation determined by ligand. We analyzed the expression of p62-ir after adrenalectomy (ADX). ADX changed the localization of GR-ir from the nucleus to the cytoplasm, but did not change the localization or immunoreactivity of p62, suggesting that nucleoporin p62 is stable regardless of intracellular signal transduction between the cytoplasm and the nucleus.

In vitro and in vivo immunocytochemistry for the distribution of mineralocorticoid receptor with the use of specific antibody.

To examine the distribution of mineralocorticoid receptor (MR) and the interactions with glucocorticoid receptor (GR) in the brain, we raised a polyclonal antibody against the transcriptional modulation domain of rat MR using the GST-fusion system. Immunoblotting analysis revealed that this antibody recognized a band with the molecular mass of MR in MR-transfected COS-1 cells and in a homogenate of rat hippocampus, and showed no cross-reactivity with GR. In vitro immunocytochemistry of both primary cultured hippocampal neurons and MR-transfected cells revealed immunoreactivity detected by this antibody in both the cytoplasm and nucleus in the absence of aldosterone (ALD), a specific agonist of MR. After 1 h of treatment with 10(-7) M ALD, the MR-immunoreactivity was accumulated in the nuclear region. In the case of GR-transfected cells, our anti-MR antibody either detected no immunopositive cells in the presence or absence of GR agonist. In our in vivo study, MR-immunoreactivity was observed in the rat hippocampus, where cell nuclei showed immunopositive reactions. These results suggest that our antibody against rat MR shows high specificity for the receptor both in liganded and unliganded forms, with no cross-reactivity to GR, and will be useful for cell biological and neuroanatomical investigations of MR.

Postnatal development of NADPH-diaphorase activity in the rat: the role of nitric oxide in the ontogeny of arginine vasopression and oxytocin.

To clarify the role of nitric oxide (NO) in the ontogeny of arginine vasopressin (AVP) and oxytocin (OXT) neurons in the hypothalamo-neurohypophysial system (HNS), we observed the coexpression pattern of NADPH-diaphorase (NADPH-d) activity and AVP- or OXT-immunoreactivity (IR) in the rat hypothalamus and posterior pituitary during the postnatal period. The enzymatic activity of NADPH-d was observed in the supraoptic nucleus (SON), paraventricular nucleus (PVN), median eminence (ME) and posterior pituitary throughout the postnatal development. AVP-containing neurons were clearly observed from postnatal day 1 in both the SON and PVN, while OXT-containing neurons were recognized from postnatal day 14. The coexistence of NADPH-d and AVP or OXT was detected in the SON from postnatal day 14. At postnatal day 21, the coexpression pattern was approximately the same as that of the SON and PVN in adult rats. Our findings indicated that the expression of NADPH-d and OXT was observed from almost the same postnatal period in both the SON and PVN. In addition, the pattern of increased numbers of NADPH-d positive fibers was similar to that of OXT-immunoreactive fibers in both the inner layer of the ME and the posterior pituitary. A good correlation was thus obtained between OXT expression and NADPH-d activity in the HNS during postnatal development. The present study suggests that NO is more closely involved in the expression and regulation of secretion of OXT than AVP.

The effects of oestrogen and progesterone on serotonin and its metabolite in the lateral septum, medial preoptic area and ventromedial hypothalamic nucleus of female rats.

The effects of sex steroid hormones on serotonin and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA) in the lateral septal nucleus (LS), the medial preoptic area (MPA) and the ventromedial nucleus of the hypothalamus (VMH) of female rats were investigated, using immunohistochemistry and high-performance liquid chromatography (HPLC). Female rats were divided into three groups: ovariectomized rats (OVX group); OVX-rats treated with estradiol benzoate alone (E2 group); and OVX-rats treated with E2 plus progesterone (E2 + P group). We analysed the density of serotonin-immunoreactive fibres with a computer-assisted image analysis system, and measured the tissue concentrations of serotonin and 5-HIAA. Many serotonin-immunoreactive fibres were observed in the LS, MPA and VMH in all three groups. The density of serotonin-immunoreactive fibres in the MPA and VMH was significantly lower in the E2 and E2+P groups compared to the OVX group, whereas the LS showed no detectable differences among the three groups. In the HPLC study, the concentrations of serotonin in the MPA and VMH of the E2 and E2+P groups were significantly lower than that in the OVX group. There was no significant difference in the concentration of serotonin in the LS. The concentration of 5-HIAA and the ratio of 5-HIAA/serotonin in the LS, MPA and VMH showed no significant differences among the OVX, E2 and E2+P groups. The present results suggest that E2 priming for sexual behaviour can affect the serotonergic system by decreasing serotonin content, but not the turnover rate, in the MPA and VMH of female rats.

The perinatal ontogeny of estrogen receptor-immunoreactivity in the developing male and female rat hypothalamus.

Developmental expression of the estrogen receptor (ER) in rat hypothalamus was examined using immunohistochemistry. In the medial preoptic nucleus and ventromedial nucleus ER-immunoreactivity was detected as early as E17, whereas ER protein expression in the periventricular preoptic nucleus and arcuate nucleus was delayed until E19. These results show that following a region specific onset of the ER protein expression sex differences in ER levels are already detectable during the perinatal period.

Role of neuropeptide Y projection on the development of serotonergic innervation in the suprachiasmatic nucleus of the rat, shown by triple intraocular grafts.

In our previous paper, the intraocular double grafts of fetal mesencephalic raphe and suprachiasmatic nucleus (SCN) demonstrated that the serotonergic fibers from raphe tissue did not show a dense innervation of SCN [28]. To examine the influence of NPY innervation from lateral geniculate nucleus (LGN) on the development of serotonergic fibers in the SCN, fetal mesencephalic raphe, SCN and LGN tissues were transplanted together into the eye chamber of adult rat. 6 weeks after transplantation, triple grafts were immunohistochemically examined. The SCN cell cluster was recognized by vasoactive intestinal polypeptide (VIP)- and arginine vasopressin (AVP)-immunoreactive neurons and The SCN cell cluster also contained a large number of serotonin-immunoreactive fibers from raphe tissue and a moderate number of neuropeptide Y (NPY)-immunoreactive fibers from LGN tissue. The present results provide information on possible NPY-serotonin interactions in the developing SCN.

Region-specific changes of tyrosine hydroxylase-immunoreactivity by estrogen treatment in female rat hypothalamus.

The effect of 17 beta-estradiol (E2) treatment for 28 days on tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the periventricular preoptic nucleus (PPN) and medial preoptic area (MPA) of ovariectomized (OVX) rats was examined by morphometric analysis. The number of TH-IR neurons in the PPN of the E2-treated group was smaller than that of the OVX group, whereas the opposite result was found in the MPA; the number of TH-IR neurons in the MPA of the E2-treated group was larger than that of the OVX group. Numerous TH-IR neurons were found in the ventromedial portion of the MPA of the E2-treated group. In both the OVX and E2-treated groups, TH-IR neurons contained many short processes up to 40 microns in length. E2 treatment caused a significant decrement of the number of neurons containing the processes in the range of 10-40 microns length in the PPN, however it caused a significant increment of the number of neurons containing the processes in the range of 5-10 microns length in the MPA. These results suggested that immunoreactivity of TH in the PPN and MPA neuron are affected by E2 treatment and that E2 might modulate the production of TH in a region-specific pattern within the hypothalamus of the female rat.

Vasopressin- and oxytocin-immunoreactive hypothalamic neurones of inbred polydipsic mice.

In the late 1950s the inbred polydipsic mice, STR/N, was discovered. The early studies indicated that the extreme polydipsia was not due to a lack of vasopressin but probably due to innate thirst of unknown origin. Because the recent investigation has revealed the presence of some functional abnormality in the brain of the STR/N mouse, we now investigated, using immunohistochemical techniques, distribution of vasopressin (AVP)- and oxytocin (OXT)-containing neurones in the hypothalamus of polydipsic strain of mouse and compared with that of the control. The pattern of distribution of AVP- and OXT-immunoreactive neurones in the paraventricular (PV), supraoptic (SO), and suprachiasmatic nuclei (SCN) of the STR/N polydipsic mouse was similar to that of the control, but the number of AVP-immunoreactive neurones was more numerous in the PVN and SON and less in the SCN in the polydipsic mouse than in the control. In addition, a discrete group of AVP- and OXT-containing neurones that was not clearly seen in the control was discovered in the STR/N. These results implicate that abnormal distribution in the brain AVP and OXT contribute to the mechanism responsible for the polydipsia shown by the strain STR/N.

[An attempt to detect genetic information by means of molecular cytochemistry].

In situ hybridization is a histochemical technique that attracts many cell biologists and others interested in developmental biology, virology, genetics and neuroendocrinology. This method gives us the precise localization and identification of individual cells which contain specific nucleic acid sequences, in a similar manner to the immunohistochemistry of cells which have a particular protein. There has been a wide range of applications for this technique. One of the most important and significant applications of in situ hybridization is the demonstration of specific mRNA in particular cells. This is quite valuable in heterogeneous tissue such as that of the hypothalamus with a various types of different cells. The combination with the immunohistochemistry enables us to study the dynamics of peptides or proteins in a certain tissue or cell. In this mini-review the logic and methodology of molecular cytochemistry, particularly, in situ hybridization, with its application in the endocrinological field was presented.

In situ hybridization histochemistry with oxytocin synthetic oligonucleotide: strategy for making the probe and its application.

A 25 mer synthetic oligonucleotide, complementary to a specific region of the oxytocin-neurophysin preprohormone messenger RNA (mRNA), was designed for its application to in situ hybridization histochemistry. The probe was 3'-end labeled with [3H] deoxycytidine triphosphate (dCTP) by using terminal deoxynucleotidyl transferase, and hybridization of the labeled probe to the mRNA in the rat hypothalamus was visualized autoradiographically. Hybridization products were specifically localized in the dorsal part of the supraoptic nucleus and the peripheral part of the paraventricular nucleus. Not only is the oligomer designed useful for distinguishing oxytocin from vasopressin gene expressing neurons, but also it is proving useful for studies of estrogen-progesterone effects on neurons in the paraventricular nucleus. Thus, these results indicate that in situ hybridization histochemistry with synthetic oligonucleotide can be a valuable approach to measuring gene expression in hypothalamic neuroendocrine cells.

Immunohistochemical demonstration of serotonin-containing nerve fibers in the hypothalamus of the monkey, Macaca fuscata.

The distributional pattern of serotonin-containing nerve fibers in the hypothalamus of the monkey (Macaca fuscata) was analyzed with the use of the peroxidase-antiperoxidase method in conjunction with a highly sensitive and specific anti-serotonin serum. The highest concentrations of serotonin-immunoreactive varicose fibers were found in the nucleus praeopticus medialis, nucleus ventromedialis hypothalami, and the complex of mammillary nuclei (nucleus praemamillaris, supramamillaris, mamillaris medialis et lateralis). However, the nucleus suprachiasmaticus, where numerous serotoninergic fibers have been reported to occur in the rat, appeared to be almost devoid of these fibers. The infundibular stalk and the intermediate and posterior lobes of the pituitary contained considerable numbers of immunoreactive fibers. The present study provides a morphological basis for possible clarification of the influence of serotoninergic projections on various neuroendocrine mechanisms in primates. Furthermore, an attempt was made to clarify the differences and similarities concerning the distributional patterns of serotoninergic nerve fibers within the monkey hypothalamus in contrast to the rat hypothalamus.

Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus.

A specific rabbit anti-CRF serum and the immunoperoxidase technique were used to show that CRF-containing neurons are mainly distributed in the paraventricular and supraoptic nuclei of the rat hypothalamus. In addition, immunoreactive neurons are scattered in other hypothalamic regions. These neurons are 20--30 micrometers in diameter. From the present and previous investigations it may be concluded that the hypothalamic magnocellular nuclei, i.e., paraventricular and supraoptic, and other hypothalamic accessory nuclei, are the producing sites not only for vasopressin and oxytocin, but also for corticotropin-releasing factor.

Immunohistochemical demonstration of serotonin nerve fibers in the hypothalamus of the cat.

Distribution of serotonin nerve fibers in the hypothalamus of the cat was studied using the peroxidase antiperoxidase (PAP) immunohistochemical method. There was a heavy concentration of serotonin nerve fibers in the nucleus suprachiasmaticus, the nucleus ventromedialis and the nucleus dorsomedialis. The distribution pattern of the serotonergic fibers in the cat was principally similar to that of the rat and monkey. However, species differences were noted in the mamillary complex, the nucleus hypothalamicus anterior, the nucleus paraventricularis and the nucleus supraopticus.