Direct hypertonic stimulation of the rat supraoptic nucleus increases c-fos expressionin glial cells rather than magnocellular neurones

We investigated whether hypertonicity acts directly on supraoptic neurones to activate c-fos expression. Hypertonic artificial cerebrospinal fluid was infused into the supraoptic nucleus (SON) via a microdialysis probe implanted 24 h previously. The rats were decapitated after 90 min for immunohistochemistry with a Fos protein antibody. Direct hypertonic stimulation increased Fos protein expression in glial cells, identified by glial fibrillary acidic protein immunoreactivity, but not in magnocellular neurones. Similarly, with in situ hybridisation c-fos mRNA expression was predominantly seen in glial cells. Fos expression in SON neurones was stimulated by systemic hypertonicity even with a microdialysis probe in the SON, and magnocellular neurones expressed Fos after direct microinjection of cholecystokinin-8S into the SON. Thus, while direct hypertonic stimulation of SON neurones activates secretion of vasopressin and oxytocin, the c-fos gene is not activated, unlike following systemic hypertonic stimulation. This indicates that excitation of neuronal electrical and secretory activity does not necessarily lead to activation of the c-fos gene. Activation of c-fos expression in glial cells by direct hypertonic stimulation may reflect their role in regulating brain extracellular fluid composition. Received: 11 March 1996 / Accepted: 24 July 1996     

Locus Coeruleus Lesions Decrease Oxytocin and Vasopressin Release Induced by Hemorrhage

The role of the noradrenergic nucleus Locus Coeruleus (LC) on hemorrhage-induced vasopressin (AVP) and oxytocin (OT) secretion was examined. Rats with LC lesion were submitted to three 1-min hemorrhage sessions at 5-min intervals; 15% of the total blood volume was withdrawn in each session. OT and AVP were measured in plasma, paraventricular (PVN) and supraoptic (SON) nuclei and in posterior pituitary (PP). LC Lesion did not affect basal plasma AVP or OT levels, but partly blocked the increase in plasma AVP and OT induced by hemorrhage. Hemorrhage produced decreases in content of AVP and OT in the PVN and SON and increased levels in the PP. These responses were attenuated in the lesioned group, but only in the PVN and PP. Data suggest a stimulatory role of the inputs from LC to PVN neurons on hemorrhage-induced OT and AVP secretion and that, this pathway is critical in the hypo-volemic neuroendocrine reflex.Special Issue Dedicated to Miklós Palkovits.     

From Punch to Profile

In this essay an aspect of my research is highlighted that has its roots in the mid seventies with the implementation of the ‘Palkovits punch’ procedure. This methodology was introduced when radiochemical and radio-immunoassay methods became sufficiently sensitive to measure key molecules for chemical neurotransmission and metabolism in minute amounts of brain tissue. For application of today’s laser guided microdissection this knowledge of functional neuro-anatomy is indispensable.Special Issue Dedicated to Miklós Palkovits.     

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.     

Organization and conservation of the GART/SON/DONSON locus in mouse and human genomes

The SON gene, which maps to human chromosome 21q22.1-q22.2, encodes a novel regulatory protein. Here we describe the organization of the Son locus in the mouse genome. The mouse Son gene spans a region of approximately 35 kb. The coding region is more than 8 kb in length and has been completely sequenced. The gene is organized into 11 coding exons and 1 noncoding 3'UTR exon, with over 70% of the coding region residing in one 5.7-kb exon. The gene contains at least one alternative exon, N/C exon 1, which can be used, by splicing, to generate a truncated form of the SON protein. Further investigation of the mouse Son locus has identified the genes directly flanking Son. The glycinamide ribonucleotide formyltransferase gene, Gart, is encoded 5' of Son in a head-to-head arrangement, with the start of both genes lying within 899 bp. Sequence comparison with the expressed sequence tagged database identified a novel gene within 65 bp of the 3' end of Son, which we have named Donson. In this unusually compact gene cluster, we have found overlap in the pattern of expression between Gart, Son, and Donson. However, at least two of these genes have very different functions. While GART is involved in purine biosynthesis, we find that SON shows the characteristics of "SR- type" proteins, which are involved in mRNA processing and gene expression.     

Exclusion of the phosphatidylinositol-specific phospholipase C β3 (PLC β3) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by hyperplasia and neoplasia in several endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a 900-kb region on chromosome 11q13. The human phosphatidylinositol-specific phospholipase C β3 (PLC β3) gene, which is located within this region, was considered to be a good candidate for the MEN 1 gene. In this study, the structure and expression of the PLC β3 gene in MEN 1 patients were investigated in more detail, to determine its potential role in MEN 1 tumorigenesis. Southern blot analysis, using blood and tumor DNA from affected persons from seven different MEN 1 families, did not reveal structural abnormalities in the PLC β3 gene. To detect possible point mutations, or other small structural aberrations, direct sequencing of PLC β3 cDNAs from two affected persons from two different MEN 1 families was performed, but no MEN 1-specific abnormalities were revealed. Several common nucleotide sequence polymorphisms were detected in these cDNAs, proving that both alleles of the PLC β3 gene were expressed and analyzed. In conclusion, these results exclude the PLC β3 gene as a candidate gene for MEN 1. Received: 20 March 1996     

Soluble Organic Nitrogen Pools in Forest soils of Subtropical Australia

Soil soluble organic N (SON) plays an important role in N biogeochemical cycling. In this study, 22 surface forest soils (0–10 cm) were collected from southeast Queensland, Australia, to investigate the size of SON pools extracted by water and salt solutions. Approximately 5–45 mg SON kg⁻¹, 2–42 mg SON kg⁻¹ and 1–24 SON mg kg⁻¹ were extracted by 2 M KCl, 0.5 M K₂SO₄ and water, on average, corresponding to about 21.1, 13.5 and 7.0 kg SON ha⁻¹ at the 0–10 cm forest soils, respectively. These SON pools, on average, accounted for 39% (KCl extracts), 42% (K₂SO₄ extracts) and 43% (water extracts) of total soluble N (TSN), and 2.3% (KCl extracts), 1.3% (K₂SO₄ extracts) and 0.7% (water extracts) of soil total N, respectively. Large variation in SON pools observed across the sites in the present study may be attributed to a combination of factors including soil types, tree species, management practices and environmental conditions. Significant relationships were observed among the SON pools extracted by water, KCl and K₂SO₄ and microbial biomass N (MBN). In general, KCl and K₂SO₄ extracted more SON than water from the forest soils, while KCl extracted more SON than K₂SO₄. The SON and soluble organic C (SOC) in KCl, K₂SO₄ and water extracts were all positively related to soil organic C, total N and clay contents. This indicates that clay and soil organic matter play a key role in the retention of SON in soil.     

Microarray Analysis of Gene Expression in the Supraoptic Nucleus of Normoosmotic and Hypoosmotic Rats

1. Hypoosmolality produces a dramatic inhibition of vasopressin (VP) and oxytocin (OT) gene expression in the supraoptic nucleus (SON). This study examines the effect of sustained hypoosmolality on global gene expression in the OT and VP magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS), in order to detect novel genes in this system that might be involved in osmoregulation in the MCNs.2. For this purpose, we used Affymetrix oligonucleotide arrays to analyze the expression of specific genes in laser microdissected rat SONs, and their changes in expression during chronic hypoosmolality. We identified over 40 genes that had three-fold or more greater expression in the SON versus total hypothalamus, and that also changed more than two fold in expression as a result of the chronic hypoosmolar treatment. These genes contained both novel as well as genes previously known to be present in the SON. All of the raw data for the genes that are expressed in the SON and altered by hypoosmolality can be found on the following NINDS website URL address: http://data.ninds.nih.gov/Gainer/Publications     

Interaction of notochord-derived fibrinogen-like protein with Notch regulates the patterning of the central nervous system of Ciona intestinalis embryos

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.     

Hypothalamic mapping of orexigenic action and Fos-like immunoreactivity following relaxin-3 administration in male Wistar rats

The insulin superfamily, characterized by common disulphide bonds, includes not only insulin but also insulin-like peptides such as relaxin-1 and relaxin-3. The actions of relaxin-3 are largely unknown, but recent work suggests a role in regulation of food intake. Relaxin-3 mRNA is highly expressed in the nucleus incertus, which has extensive projections to the hypothalamus, and relaxin immunoreactivity is present in several hypothalamic nuclei. In the rat, relaxin-3 binds and activates both relaxin family peptide receptor 1, which also binds relaxin-1, and a previously orphaned G protein-coupled receptor, RXFP3. These receptors are extensively expressed in the hypothalamus. The aims of these studies were twofold: 1) map the hypothalamic site(s) of the orexigenic action of relaxin-3 and 2) examine the site(s) of neuronal activation following central relaxin-3 administration. After microinjection into hypothalamic sites, human relaxin-3 (H3; 180 pmol) significantly stimulated 0- to 1-h food intake in the supraoptic nucleus (SON), arcuate nucleus (ARC), and the anterior preoptic area (APOA) [SON 0.4+/-0.2 (vehicle) vs. 2.9+/-0.5 g (H3), P<0.001; ARC 0.7+/-0.3 (vehicle) vs. 2.7+/-0.2 g (H3), P<0.05; and APOA 0.8+/-0.1 (vehicle) vs. 2.2+/-0.2 g (H3), P<0.05]. Cumulative food intake was significantly increased相似文献   

Urocortin 1 administered into the hypothalamic supraoptic nucleus affects open-field behaviour in rats

The presence of both Urocortin 1 (Ucn1) and corticotropin-releasing factor 2 receptors (CRF₂R) in the hypothalamic supraoptic nucleus (SON) suggests that endogenous Ucn1 released within this brain area acts as a local signal that might be involved in the regulation of not only endocrine but also behavioural stress responses. To test this hypothesis, we monitored the effects induced by the administration of a range of doses of synthetic Ucn1 (0.001–1.0 μg) bilaterally into the SON of rats in the open field test (OFT). Ucn1 administration produced an inverted U-shaped dose–response curve on OFT behaviour, in particular the dose of 0.01 μg of Ucn1 significantly increased the number of rearing and grooming episodes without affecting locomotion. In addition, this dosage augmented also the latency to visit the centre of the open field. Pre-treatment with the CRF₂R antagonist, astressin-2B (0.1 μg) normalized Ucn1 treatment-induced effects. These results suggest that Ucn1 released within the SON area interacts with CRF₂R to control the state of arousal.     

Clathrin-mediated endocytic signals are required for the regeneration of, as well as homeostasis in, the planarian CNS

Planarians have a well-organized central nervous system (CNS), including a brain, and can regenerate the CNS from almost any portion of the body using pluripotent stem cells. In this study, to identify genes required for CNS regeneration, genes expressed in the regenerating CNS were systematically cloned and subjected to functional analysis. RNA interference (RNAi) of the planarian clathrin heavy chain (DjCHC) gene prevented CNS regeneration in the intermediate stage of regeneration prior to neural circuit formation. To analyze DjCHC gene function at the cellular level, we developed a functional analysis method using primary cultures of planarian neurons purified by fluorescence-activated cell sorting (FACS) after RNAi treatment. Using this method, we showed that the DjCHC gene was not essential for neural differentiation, but was required for neurite extension and maintenance, and that DjCHC-RNAi-treated neurons entered a TUNEL-positive apoptotic state. DjCHC-RNAi-treated uncut planarians showed brain atrophy, and the DjCHC-RNAi planarian phenotype was mimicked by RNAi-treated planarians of the mu-2 (micro2) gene, which is involved in endocytosis, but not the mu-1 (micro1) gene, which is involved in exocytosis. Thus, clathrin-mediated endocytic signals may be required for not only maintenance of neurons after synaptic formation, but also axonal extension at the early stage of neural differentiation.     

Early embryonic development of the central nervous system in the Australian crayfish and the Marbled crayfish (Marmorkrebs)

This study sets out to provide a systematic analysis of the development of the primordial central nervous system (CNS) in embryos of two decapod crustaceans, the Australian crayfish Cherax destructor (Malacostraca, Decapoda, Astacida) and the parthenogenetic Marbled crayfish (Marmorkrebs, Malacostraca, Decapoda, Astacida) by histochemical labelling with phalloidin, a general marker for actin. One goal of our study was to examine the neurogenesis in these two organisms with a higher temporal resolution than previous studies did. The second goal was to explore if there are any developmental differences between the parthenogenetic Marmorkrebs and the sexually reproducing Australian crayfish. We found that in the embryos of both species the sequence of neurogenetic events and the architecture of the embryonic CNS are identical. The naupliar neuromeres proto-, deuto-, tritocerebrum, and the mandibular neuromeres emerge simultaneously. After this “naupliar brain” has formed, there is a certain time lag before the maxilla one primordium develops and before the more caudal neuromeres follow sequentially in the characteristic anterior–posterior gradient. Because the malacostracan egg-nauplius represents a re-capitulation of a conserved ancestral information, which is expressed during development, we speculate that the naupliar brain also conserves an ancestral piece of information on how the brain architecture of an early crustacean or even arthropod ancestor may have looked like. Furthermore, we compare the architecture of the embryonic crayfish CNS to that of the brain and thoracic neuromeres in insects and discuss the similarities and differences that we found against an evolutionary background.