Detection of TRH extended peptides in rat hypothalamus using an antibody raised to pGluHisProGlyLys

An antibody was raised to the synthetic pentapeptide pGluHisProGlyLys which, in radioimmunoassay (RIA), could detect the pentapeptide at a level of 10 fmole per tube and exhibited <0.5 per cent cross reactivity with a series of related peptides. The RIA was used to demonstrate the presence of C-terminally extended forms of thyrotropin releasing hormone (TRH) in rat hypothalamus. After extraction, the endogenous peptides were resolved by gel exclusion chromatography and TRH-extended peptides were revealed by trypsin digestion to release the pentapeptide. The TRH extended peptides occurred in substantial quantity, approximately 11 pmoles/g, indicating that only partial processing of the gene duplicated prohormone takes place.     

CRF-containing neurons of the rat hypothalamus

Summary The immunoreactive CRF-neurons of the rat hypothalamus have been examined immunohistochemically employing anti-rat CRF serum. These neurons are confined to the paraventricular nucleus, dorsomedial-lateral hypothalamic area, and suprachiasmatic nucleus, and are, respectively, also immunoreactive to anti-Met-enk, -alpha-MSH, and -VIP sera. Intraventricular administration of colchicine (50 g/5 l/rat) induces a dramatic enhancement of the immunostainability of the cell somata, and also accelerates the development of immunoreactivity of other stored peptides, especially in the paraventricular nucleus.The CRF-neurons respond to adrenalectomy by showing increased immunoreactivity and an increase in the number of cell bodies; in the dorsomedial-lateral area and suprachiasmatic nucleus, there is also an enhanced immunoreactivity for alpha-MSH and VIP, respectively. CRF-cells in the paraventricular nucleus become markedly hypertrophied, but do not show any enhanced immunoreactivity for Met-enk. Since the axons of the paraventricular neurons run to the median eminence, it is probable that they are involved with the endocrine control of hypophysial ACTH release. It is concluded that the CRF-containing neurons in rat hypothalamus consist of three types which are functionally and morphologically different.     

A retinal projection to the lateral hypothalamus in the rat

Summary This study presents evidence for a retinal projection to neurons in the lateral hypothalamic area (LHA) of the albino rat. In Golgi-Kopsch material dendrites from LHA-neurons are observed to extend through the supraoptic commissures into the optic tract. The presence of dendrites in the optic tract is confirmed by electron microscopy. Numerous axon terminals are observed forming asymmetric synaptic contacts with these dendritic profiles. Following bilateral enucleation, many of the preterminal axons and terminals in synaptic contact with dendrites in the optic tract demonstrate dark degeneration. After intraocular injection of horseradish peroxidase, there is marked labeling of preterminal axons and terminals in the optic tract. These observations indicate that LHA neurons receive a direct retinal projection from terminals making synaptic contact with dendrites of LHA-neurons extending into the optic tract.     

A group of neurons highly reactive for enkephalins in the rat hypothalamus

Enkephalin-like immunoreactivity was demonstrated in a group of highly reactive neurons (HRN) of the rat hypothalamus by the biotin-avidin immunohistochemical technique. The location of the HRN spans several nuclei but the consistent immunoreactivity, the constant topography, and the uniform dimensions of the neurons suggest that they belong to one group. At its caudal end the group appears within the rostral dorso-medial nucleus. At the level of the caudal paraventricular nucleus (PVH) the HRN are assembled in a spherical pattern around a subgroup of neurons in the anterior hypothalamus (AH). The HRN then are found in a position directly between the PVH and the fornix. Before the HRN disappear at the level of the caudal preoptic area, many of the HRN become associated with the fornix. The close association of the HRN with the AH subgroup and the fornix suggests that the HRN may influence the activity of these structures. The HRN are small to medium in size and their short processes suggest that the HRN communicate with other neurons in their vicinity. The areas of the hypothalamus in which the HRN are found are involved in neuroendocrine and thermoregulatory functions suggesting that the HRN may play a role in modifying the activity of neurons and fibres involved in these functions.     

Ontogenetic appearance of immunoreactive GRF-containing neurons in the rat hypothalamus

Summary Ontogenetic development of GRF-containing neurons in the rat hypothalamus was studied employing antisera which were generated against hpGRF (1–44)NH₂ and rhGRF(1–43)OH: anti-hpGRF-C and -rhGRF sera recognize the species-specific C-terminal portions of the peptides, and anti-hpGRF-MC and -N sera recognize hpGRF(27–44)NH₂ and the N-terminal portion of hpGRF(1–44)NH₂, respectively. The anti-hpGRF-C and-rhGRF sera stained different neuronal cell bodies, which were localized in distinct hypothalamic areas. The former serum did not stain the axonal terminals in the median eminence, but the latter stained them strongly. The antihpGRF-MC and -N sera stained neuronal cell bodies, some of which corresponded to those immunolabelled with antihpGRF-C or -rhGRF serum. The anti-rhGRF serum first demonstrated immunoreactive perikarya in the ventral-lateral border of the arcuate nucleus of 19.5-day-old fetuses that had received an intraventricular colchicine administration 24 h previously. The immunoreactive fibers were recognized first in the external layer of the median eminence of untreated fetuses on day 19.5 of gestation, and then they increased in amount with development. No immunore-active fibers, however, were found in the median eminence of colchicine-treated animals during the fetal period. It is concluded that in rats GRF may be synthesized in the perikarya on day 18.5 of gestation and conveyed to the median eminence without delay via axonal flow.     

Substance P-related peptides in the hypothalamus of Amphibia

Summary The occurrence of Substance P-(SP)-related peptides in the hypothalamus of three species of Amphibia (newt, clawed, toad, frog) was studied immunohistochemically employing the indirect immunofluorescence method or a double-step technique (indirect immunofluorescence followed by the peroxidase-antiperoxidase complex method). SP-like immunopositive fibers are seen throughout the hypothalamus. They are especially abundant in the preoptic area and in the outer zone of the median eminence, suggesting a role of SP-related peptides in the hypothalamo-hypophysial regulation in these animals. Some SP-like neurons are seen in the posterior hypothalamus and in the preoptic area. In the newt, such SP-like immunopositive neurons occur frequently in the preoptic periventricular grey.Work performed under the C.N.R. project Biologia della Riproduzione     

The location of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence

Summary The location of the perikarya of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence were studied by immunohistochemistry and radioimmunoassay after placing stereotaxic electrolytic lesions in several parts of the hypothalamus. The principal location of the cell somata was found to be in the ventral part of the medial preoptic area; their pathways were classified into a main baso-lateral pathway and an accessory descending pathway branching off from the former. The main pathway was found to cross in the vicinity of the corresponding neuronal perikarya. The central median eminence and the dorsal and ventral walls of the tubero-infundibular sulcus of the caudal part of the median eminence are innervated mainly by the baso-lateral pathway. On the other hand, the rostral and most caudal portions of the median eminence are innervated principally by the descending pathway and have a subsidiary dual innervation. The projection of LH-RH neurons to the OVLT is believed to originate from perikarya adjacent to this circumventricular organ.This work was supported in part by a grant (No. 248093, 321426) from the Ministry of Education, Science and Culture, Japan     

Substance P degrading systems of rat parotid and hypothalamus

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu⁶]substance P6–11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K⁺ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu⁶]substance P6–11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe⁸-Gly⁹ with minor cleavage sites between Phe⁷-Phe⁸ and Gly⁹-Leu¹⁰. In the rat parotid slice system the major cleavage occurs between Gly⁹-Leu¹⁰ with a minor cleavage site between Phe⁷-Phe⁸. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH₂, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

Plant antigens cross-react with rat polyclonal antibodies against KLH-conjugated peptides

Keyhole limpet hemocyanin (KLH)-conjugated peptides are routinely used to raise polyclonal antibodies for biochemical or immunolocalization studies. Rats are suitable for producing antisera against plant antigens as they often lack non-specific response towards plant materials. We attempted to obtain rat antisera against peptides derived from several plant proteins. However, most antisera recognized the same background KLH-related plant antigen (KRAP) in Arabidopsis and tobacco. We characterized KRAP with respect to size and cellular localization and examined possible antigen-specific reasons for the failure of most immunizations. We also found no reports of successful use of rat anti-KLH-peptide antibodies in plant studies. We thus believe that the rat-KLH:peptide system is poorly suited for production of antibodies, especially against plant antigens, and should be used with caution, if at all.     

Ultrastructural localization of the receptor for leptin in the rat hypothalamus

Ultrastructural localization of the leptin receptor in the rat hypothalamus was studied by immunocytochemistry. The antiserum against the leptin receptor which was used specifically recognized the carboxy terminal of the cytoplasmic domain. Intense leptin receptor immunoreactivity was detected in the arcuate, paraventricular, and ventromedial nuclei of the hypothalamus and in the lateral hypothalamic area. At the ultrastructural level, leptin receptor-like immunoreactivity appeared to be concentrated predominantly in perikarya and dendrites of these areas and strong immunolabeling for the leptin receptor was detected in the plasma membrane, rough endoplasmic reticulum, Golgi apparatus, and cytoplasmic matrix. This study provides the first detailed fine structure of leptin receptor-immunoreactive neurons in the rat hypothalamus. It may help to provide better understanding of the functions of leptin in the rat hypothalamus.     

Circadian and sleep-deprivation variations of monophosphorylated MAP-Kinase in hypothalamus and pons of rats

Behavior and physiological changes are under the influence of circadian and homeostatic variations. Temporal alignment regulates timing of neurobiological phenomena, such as protein phosphorylation. In the current report, we describe the circadian and sleep homeostatic phosphorylated mitogen-activated protein kinase (MAP-K) variations in hypothalamus and pons of rats across 24 h as well as after sleep deprivation. In the circadian study, MAP-K expression showed a building-up profile during the dark phase in hypothalamus, whereas an increase across the lights-on period was found in pons. On the other hand, that phosphorylation of MAP-K in hypothalamus and pons displayed a significant reduction after sleep rebound period. Data demonstrate that MAP-K phosphorylation undergoes circadian and sleep homeostatic variations in brain areas linked to sleep modulation.     

Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus

Summary The rat hypothalamus was studied at the light microscopic level with the use of single and double immunocytochemical staining methods. It was shown that the rat supraoptic and paraventricular hypothalamic nuclei, and their accessory neurosecretory nuclei, do not contain magnocellular somatostatin neurons. The distribution of the hypothalamic parvocellular somatostatin cells is described. The parvocellular component of the rat hypothalamic paraventricular nucleus is, at least partly, composed of somatostatin cells: they form a fairly well circumscribed periventricular cell mass. The rat suprachiasmatic nuclei contain separate somatostatin neurons and vasopressin neurons. Scattered somatostatin cells are present in the entire arcuate nucleus. In addition to the periventricular somatostatin cells located in the preopticanterior hypothalamic area and in the arcuate nucleus, the rat hypothalamus also contains numerous scattered somatostatin cells located distant from the third ventricle.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Intranuclear inclusions in pericytes of the hypothalamus of the rat

Summary This paper deals with the ultrastructure of two types of intranuclear inclusions, nuclear bodies and membranous lamellar bodies, present in hypothalamic pericytes of intact adult rats. The nuclear bodies exhibited simple and granular forms, whereas the membranous lamellar bodies were entirely made up of myelin-like membrane whorls.The occurrence of these bodies in nuclei of pericytes has never been previously reported. The origin and functional meaning of such structures is discussed in the light of recent ultrastructural and biochemical studies on nuclear inclusions.     

Intragranular co-storage of neuropeptide Y and arginine vasopressin in the paraventricular magnocellular neurons of the rat hypothalamus

Summary Certain populations of arginine vasopressin (AVP) neurons in the magnocellular paraventricular nucleus became immunoreactive for neuropeptide Y (NPY) when rats were treated with colchicine or monosodium glutamate (MSG). The co-storage of these peptides was examined by empooying a post-embedding electron-microscopic immunohistochemistry technique using goldlabeled antibodies to the two peptides. In colchicinetreated rats, the neuronal perikarya contained numerous secretory granules showing co-storage of the two peptides. The cells of the MSG-treated rats were characterized by having well-developed Golgi bodies with the granular structures also co-storing the two peptides, although the secretory granules in the perikarya were rather fewer than in the colchicine-treated rats. It is concluded that the destruction of the arcuate nucleus by MSG-treatment may potentiate the synthesis of NPY in AVP neurons, the synthesis of which is latent in intact animals.     

Orexigenic action of oral zinc: metabolomic analysis in the rat hypothalamus

ABSTRACT

We previously reported an orexigenic action of oral zinc administration in male Sprague-Dawley (SD) rats during an early stage of feeding with a zinc-deficient diet, without decreased zinc concentrations in tissues. The overall conclusion was that orally but not intraperitoneally administered zinc stimulates food intake in short-term zinc-deficient-diet fed rats. We here investigate the mechanism of the orexigenic action of zinc using GC-MS/MS-targeted metabolomic analysis in the rat hypothalamus. Four-week-old, male SD/Slc rats were used, and after 2 days of feeding with a zinc-deficient diet, 3 mg of ZnSO₄ in 5 mL saline solution were administered to each rat either orally or intraperitoneally. Three hours after administration, the rats were sacrificed and the hypothalamus were excised and analyzed. We found that the oral administration group showed increased concentrations of 3-aminopropanoic acid (β-alanine), hypotaurine, dopamine, and biotin. In light of metabolomic analysis of these results, we indicate directions for further research.     

Ultrastructural localization of radiolabelled L-dopa in the endocrine hypothalamus of the rat

Summary Light and electron microscopic autoradiography has been employed to define the neuroanatomical patterns of uptake and binding of radiolabelled L-dopa in the endocrine hypothalamus of the rat. A dorsomedial continuum of arcuate and periventricular neurons selectively sequester ³H L-dopa 20 min following its intraventricular infusion. By 40 and 60 min following the infusion labelling of neurons is minimal and supports the notion of rapid degradation. Other cell compartments such as tanycytes demonstrate uptake of ³H L-dopa. The ultrastructural localization and distribution of radiolabelled L-dopa (or its metabolites) in the rodent hypothalamus is discussed with respect to mechanisms and cell compartments involved in neuroendocrine regulatory processes.Supported by USPHS Program Project Grant NS-11642-04 (DES) and RR-05403Career Development Awardee RO4GM-70001     

Somatostatin concentration and binding in the rat hypothalamus and striatum during severe insulin-induced hypoglycemia

Hypoglycemia was induced by administration of insulin (40 I.U./kg) to 24 h fasted rats. Somatostatinlike immunoreactivity (SLI) and¹²⁵I-Tyr¹¹-somatostatin binding were measured in the striatum and hypothalamus at the onset of hypoglycemic coma (5–10 min). No significant changes in SLI concentration were detected in either site although the total number of specific somatostatin receptors in the striatum membranes, but not in the hypothalamus, decreased in insulin-injected rats when compared with the control group.     

Ceruletide acts in the abdomen, not in the brain, to produce satiety

Ceruletide (caerulein), a decapeptide extracted from the skin of the frog, Hyla caerulea, is very similar in structure to the C-terminal octapeptide of cholecystokinin (CCK-8). Although ceruletide and CCK-8 act through similar or identical receptors to produce the same visceral effects, previous studies in the rat suggested that peripherally administered ceruletide acted directly on the ventromedial hypothalamic (VMH) area to decrease food intake, but peripherally administered CCK-8 acted at a vagally innervated abdominal site to decrease food intake. Since it is unprecedented for these two peptides to produce the same effect by acting at different sites, we investigated the site of action of ceruletide's satiety effect in the rat and compared it to the site of action of CCK-8. The major results were: (1) intraperitoneal administration of ceruletide and CCK-8 inhibited food intake, but intraventricular administration did not; (2) the satiety effect of ceruletide and CCK-8 was not changed by bilateral lesions of the VMH; and (3) the satiety effect of ceruletide and CCK-8 was abolished or markedly reduced by bilateral abdominal vagotomy. We conclude that ceruletide acts at the same vagally innervated abdominal site to produce satiety as CCK-8 does and that neither peptide acts directly on the VMH area.