Immunohistochemical localization in the rat brain of the precursor for thyrotropin-releasing hormone.

A rabbit antiserum to a peptide sequence present in the precursor for thyrotropin-releasing hormone (proTRH), deduced from cloned amphibian-skin complementary DNA, was raised by immunization with the synthetic decapeptide Cys-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-Cys (proTRH-SH). Immunohistochemical studies on rat brain tissue showed staining of neuronal perikarya in the parvicellular division of the paraventricular nucleus of the hypothalamus and the raphe complex of the medulla, identical to that already described for thyrotropin-releasing hormone (TRH). Immunostaining was abolished by preincubation with proTRH-SH (10(-6)M) but not TRH (10(-5)M). Both TRH precursor and TRH were located in neurons of the paraventricular nucleus. However, in contrast to the findings for TRH, no staining was observed in axon terminals of the median eminence. These results suggest that a TRH precursor analogous to that reported in frog skin is present in the rat brain and that TRH in the mammalian central nervous system is a product of ribosomal biosynthesis.

Immunoreactive vasopressin and oxytocin: concentration in individual human hypothalamic nuclei.

Individual hypothalamic nuclei were microdissected from brain tissue of ten human subjects who had died suddenly while in apparent good health. Appreciable amounts of vasopressin and oxytocin immunoreactivity were found by specific radioimmunoassay in six hypothalamic nuclei including supraoptic and paraventricular nuclei. Vasopressin and oxytocin are presumed to be synthesized in supraoptic and paraventricular nuclei for axonal transport to the posterior pituitary for storage and release. Vasopressin and oxytocin in other hypothalamic nuclei may be a part of this system of neurosecretion or may serve some other function.

Estradiol is concentrated in tyrosine hydroxylase-containing neurons of the hypothalamus.

Localization of [3H]estradiol in tyrosine hydroxylase-containing neurons of rat brain was shown by a combined technique of autoradiography and immunohistochemistry. [3H]Estradiol was concentrated in the nuclei of tyrosine hydroxylase-containing neurons in the nucleus arcuatus, nucleus periventricularis hypothalami, and the zona incerta. These results suggest that estradiol acts directly on dopamine-producing neurons of the tuberoinfundibular system and incertohypothalamic system.

Endothelin: a novel peptide in the posterior pituitary system.

Endothelin (ET), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, is present in the porcine spinal cord and may act as a neuropeptide. Endothelin-like immunoreactivity has now been demonstrated by immunohistochemistry in the paraventricular and supraoptic nuclear neurons and their terminals in the posterior pituitary of the pig and the rat. The presence of ET in the porcine hypothalamus was confirmed by reversed-phase high-pressure liquid chromatography and radioimmunoassay. Moreover, in situ hybridization demonstrated ET messenger RNA in porcine paraventricular nuclear neurons. Endothelin-like immunoreactive products in the posterior pituitary of the rat were depleted by water deprivation, suggesting a release of ET under physiological conditions. These findings indicate that ET is synthesized in the posterior pituitary system and may be involved in neurosecretory functions.

Suprachiasmatic nucleus vasopressin messenger RNA: circadian variation in normal and Brattleboro rats.

In situ hybridization of an oligonucleotide probe complementary to vasopressin messenger RNA (mRNA) in sections from normal or Brattleboro rat hypothalami revealed hybridization densities in each of three vasopressin-rich nuclei: the supraoptic, paraventricular, and suprachiasmatic. When entrained to a daily light-dark cycle, each rat strain displayed diurnal variation in hybridizable mRNA in the suprachiasmatic, but not in the supraoptic or paraventricular nuclei. The higher values for suprachiasmatic mRNA in the morning correlate well with previously elucidated morning increases in vasopressin immunoreactivity in the cerebrospinal fluid. These results support the utility of in situ hybridization techniques for elucidating physiological influences on regional peptidergic function, are consistent with a prominent role for vasopressinergic suprachiasmatic neurons in generating the cerebrospinal fluid vasopressin rhythm, and suggest that regulation of this mRNA rhythm is not dependent on release of intact peptide.

Oxytocin content of microdissected areas of rat hypothalamus.

Oxytocin content has been measured by radioimmunoassay in microdissected hypothalamic nuclei. Equal concentrations of oxytocin were found in the supraoptic and the paraventricular nuclei, indicating that both are major sources of the hormone. The concentration of oxytocin in the median eminence was more than three times that in either the supraoptic or the paraventricular nuclei, and significant amounts of oxytocin were also found in the arcuate nucleus and in tow anterior hypothalamic nuclei.

On the origin of Leu-enkephalin and Met-enkephalin in the rat neurohypophysis.

The posterior lobe of the pituitary contains large amounts of Leu- and Met-enkephalin (LE and ME, respectively). A marked depletion of ME (81.9%) and LE (94.5%) in the posterior pituitary occurred after transection of the pituitary stalk. This indicates that most, if not all, of the enkephalins are in processes of central neurons. In the present study, I attempted to determine the source(s) of the LE- and ME-containing fibers in the posterior pituitary by examining the effects of hypothalamic lesions or fiber transections on the LE and ME levels. Lesions of the hypothalamic paraventricular nuclei caused ME and LE levels in the posterior pituitary to decrease significantly (55.6% and 27.6%, respectively). Deafferentation of the medial basal hypothalamus (creating islands of tissue containing the ventromedial and arcuate nuclei) resulted in a marked reduction in LE (94.1%) and ME (54.7%). Treating neonatal rats with monosodium glutamate resulted in a selective destruction of arcuate nucleus neurons, but did not affect LE and ME concentrations in the posterior pituitary. Thus, about half of the ME in the posterior pituitary seems to be provided by neurons in the vicinity of the paraventricular and ventromedial nuclei, whereas only about one quarter of the LE in the posterior pituitary is in processes of the paraventricular nucleus neurons. The remainder of the LE is contributed to the posterior pituitary by neurons outside the medial basal hypothalamus, probably by the supraoptic nucleus neurons. These findings are consistent with the hypothesis that LE and ME may be localized in separate populations of nerve endings in the neurohypophysis and may have different roles.

Biochemical and immunochemical studies of supraoptic and paraventricular cultures.

A tissue culture model was established for the study of hypothalamic peptide synthesis and secretion. Microdissected explants of the paraventricular and supraoptic regions from Sprague-Dawley rats (neonates or young rats) were maintained in culture for up to 3 weeks. Studies were performed to evaluate vasopressin and oxytocin content (medium and tissue levels), immunocytochemical localization, and biosynthetic activity. Immunocytochemical staining for oxytocin, neurophysin, and neuron-specific enolase showed positive neurons in both the paraventricular and supraoptic cultures. In many cases, the neurons were large (30-40 microns) and bipolar, resembling the classic magnocellular neuron. Measurement of tissue and medium content showed the continued presence of vasopressin and oxytocin in the cultured explants. Even after 3 weeks, there were significant amounts of vasopressin present. Biosynthesis was evaluated by determining the incorporation of 35S-labeled cystine or cysteine into proteins and peptides. The medium and tissue extracts were separated by reverse-phase high-performance liquid chromatography. Results showed that most of the labeled peptides were released into the medium rather than stored. There were two labeled peaks in the medium, which chromatographically resembled native vasopressin and oxytocin. Treatment with a protein synthesis inhibitor, either puromycin or cycloheximide, resulted in a decrease in labeled peptides. A comparison of 35S-labeled cystine and cysteine showed that the latter was the label of choice, with significantly greater incorporation.

Distributions of pro-vasopressin expressing and pro-vasopressin deficient CRH neurons in the paraventricular hypothalamic nucleus of colchicine-treated normal and adrenalectomized rats.

The corticotropin-releasing hormone (CRH) neurosecretory system in normal rats consists of two major subpopulations of parvicellular neurons in the hypothalamic paraventricular nucleus distinguished by the presence or absence of coexistent vasopressin precursor (pro-AVP)-derived peptides. These neurons project to the external zone of the median eminence, where the two subtypes of axons (CRH +/AVP + and CRH+/AVP-) were previously found to be approximately equal in number. The present study was undertaken 1) to determine whether the relative numbers of pro-AVP expressing and pro-AVP deficient perikarya in the paraventricular nucleus corresponded to what we previously found for the axons in the median eminence, 2) to map the two cell types throughout the entire paraventricular nucleus to determine whether significant differences existed in their distributions, and 3) to ascertain whether or not the pro-AVP deficient subpopulation expressed pro-AVP after adrenalectomy. Postembedding electron microscopic immunocytochemistry on serial ultrathin sections was used to identify the peptide phenotypes of perikarya in the paraventricular nucleus in normal rats and 7 days after adrenalectomy with and without colchicine treatment. The peptide phenotypes of neuronal perikarya in the paraventricular nucleus were identified by using antibodies to CRH, AVP, neurophysin (NP), the C-terminal glycopeptide of pro-AVP (GP), and oxytocin-associated neurophysin (NPOT). Groups of three serial coronal ultrathin sections were analyzed at 200-micron intervals throughout the entire rostrocaudal extent of the paraventricular nucleus. The sections in each group were stained for CRH, a pro-AVP-derived peptide (AVP, NP, or GP), and NPOT, respectively. Parvicellular CRH neurons were defined as CRH-positive cells, approximately 10 micron in diameter, that did not contain detectable NPOT. Pro-AVP expressing cells were defined as staining positively for AVP, GP, or NP and negatively for NPOT. Approximately equal numbers of pro-AVP expressing ("NPAVP+") and pro-AVP deficient ("NPAVP-") parvicellular CRH neurons were found within the paraventricular nucleus of colchicine-treated normal rats, and the two subtypes were distributed differently within the paraventricular nucleus. Although the pro-AVP expressing CRH cells stained intensely for NP and GP, staining for AVP was quite variable and difficult to quantify in colchicine-treated normal animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of peptide histidine-isoleucine/vasoactive intestinal polypeptide-immunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor- and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus.

The distribution of peptide histidine-isoleucine (PHI) and vasoactive intestinal polypeptide (VIP), two peptides derived from the same precursor molecule, was analysed with immunohistochemistry in the central nervous system of the rat, and to a limited extent in some other species including sheep, monkey and man. Special attention was focused on possible cross-reactivity between PHI antisera and corticotropin releasing factor in parvocellular neurons in the hypothalamic paraventricular nucleus projecting to the external layer of the median eminence. (1) Characterization of the PHI and VIP antisera revealed that they recognized different sequences of the peptide molecules. One of the PHI antisera (PHI-N), although mainly N-terminally directed, also probably contained an antibody population directed against the C-terminal amino acid in PHI which is an amidated isoleucine. Rat and human corticotropin releasing factor but not ovine also have an amidated isoleucine in C-terminal position. (2) PHI- and VIP-like immunoreactivity were found with parallel and overlapping distribution in all areas investigated in the rat central nervous system. In many cases coexistence of the two immunoreactivities could be directly demonstrated. PHI neurons were found in some areas so far not know to contain PHI/VIP neurons, including the dorsal septum, the septofimbrial nucleus, the stria terminalis and lamina V of the spinal cord. (3) Using an antiserum directed against the amino acid sequence 111-122 of the VIP/PHI precursor, immunoreactive cell bodies were seen in some areas containing VIP and PHI neurons. PHI- and VIP-like immunoreactivity were expressed in parallel in increasing amounts in the superficial laminae of the dorsal horn after transection of the sciatic nerve [G. P. McGregor et al. (1984) Neuroscience 13, 207-216; S. A. S. Shehab and M. E. Atkinson (1984) J. Anat. 139, 725; S. A. S. Shehab and M. E. Atkinson (1986) Expl Brain Res. 62, 422-430]. (5) The PHI-N antiserum stains large numbers of immunoreactive cells in the parvocellular part of the paraventricular nucleus and these cells are mostly identical with corticotropin releasing factor-positive neurons. Absorption experiments suggested that this PHI-N-like immunoreactivity to a large extent represented cross-reactivity with rat CRF and that earlier demonstration of many PHI-positive neurons in the paraventricular nucleus probably represents an artefact as proposed by F. Berkenbosch et al. (Neuroendocrinology 44, 338-346). However, some cells did, in fact, contain VIP- as well as PHI-like immunoreactivity as was shown with antisera not cross-reacting with corticotropin releasing factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural identification of noradrenergic nerve terminals and vasopressin-containing neurons of the paraventricular nucleus in the same thin section.

Since many peptidergic cell groups receive a diverse and complex monoaminergic innervation, we have developed a double-label procedure to visualize a peptide and a catecholamine in the same ultrathin section. Radiolabeled norepinephrine (NE) is applied locally and its reuptake into NE terminals is demonstrated by ultrastructural radioautography. Controls in this and other studies demonstrate that the NE labels only NE (and possibly epinephrine) terminals and not dopaminergic or serotonergic terminals. In the same tissue, vasopressin is localized by immunocytochemistry on unembedded sections that are subsequently embedded in epoxy resins for thin sectioning. The procedure as described here shows that NE terminals in the periventricular zone of the paraventricular nucleus of the hypothalamus innervate both vasopressin-positive and vasopressin-negative structures. This technique is useful in determining the chemical connectivity of the hypothalamus.

Analysis of vasopressin gene expression by in situ hybridization and immunohistochemistry in semi-thin sections.

We have designed a procedure to investigate vasopressin (AVP) gene expression on plastic-embedded tissue by using in situ hybridization to detect AVP mRNA and immunohistochemistry to detect AVP. Rat brain was fixed and vibratome slices were incubated with a 45-base synthetic oligonucleotide complementary to AVP mRNA labeled with 35S, embedded in Araldite, and cut into semi-thin serial sections that were either processed for autoradiography or treated with an AVP antiserum. The results show that AVP mRNA is detectable in magnocellular neurons of the supraoptic and paraventricular nuclei in both vibratome and semi-thin sections. Osmication after hybridization does not modify the signal. AVP mRNA is restricted to the cytoplasm of magnocellular neurons and to the proximal portion of certain processes. Neurons labeled with the AVP probe were also stained with the AVP antiserum. AVP mRNA quantity and the intensity of AVP immunoreactivity are not consistently related in neurons. At least two hypotheses must be considered to explain these differences: first, the procedure presently used could lead to a reaction intensity that does not exactly reflect the amount of antigen or mRNA present in cells; second, the difference observed may reflect the fact that transcriptional and translational events are not constantly linked and can be regulated differently from one AVP neuron to another. This method provides a way to detect mRNA on semi-thin sections together with antigenic molecules and to accurately investigate gene expression in complex tissues with optimal histological quality.

A monoclonal antibody to vasopressin: preparation, characterization, and application in immunocytochemistry.

The hypothalamo-neurohypophysial system, containing the hormones oxytocin (OT) and vasopressin (VP) and their associated carrier proteins, the neurophysins (NPS), has been the subject of extensive investigation for more than 40 years. This system has been reinvestigated during the last decade by application of immunocytochemical methods employing the rabbit antisera to the hormones and NPS. In this study we describe the preparation and characterization of a monoclonal antibody to VP and its application in immunohistochemistry. The antibody did not cross-react with OT or arginine vasotocin (AVT). Its antigenic determinants as characterized by absorption with various VP analogs included two aromatic amino acids: Phe in position 3, and to a lesser extent Tyr in 2. Tissue fixation with formaldehyde resulted in inadequate immunostaining as compared to glutaraldehyde, most likely due to interference with the aromatic amino acid determinants by the former fixative.

Vasopressin gene expression in the normal and Brattleboro rat: a histological analysis in semi-thin sections with biotinylated oligonucleotide probes.

We analyzed expression of the vasopressin (AVP) gene in semi-thin sections in normal and Brattleboro rats by using in situ hybridization and immunohistochemistry. AVP mRNA was detected as follows: vibratome sections of rat hypothalamus were hybridized with a biotinylated oligonucleotide probe, embedded in Araldite, and cut into semi-thin sections which were reacted with streptavidin-alkaline phosphatase and the appropriate substrate. Adjacent serial sections were treated by immunohistochemistry to detect AVP or oxytocin immunoreactivity. In normal rat, AVP mRNA can be detected in magnocellular neurons of the supraoptic and paraventricular nuclei and in parvocellular neurons of the suprachiasmatic nucleus. AVP mRNA was present throughout the cytoplasm of the cell bodies, their processes, and in punctate structures in the vicinity of the AVP cell bodies. Most neurons containing AVP mRNA also contain AVP immunoreactivity, but the staining intensity was not consistently correlated for each reaction. A few neurons contained AVP mRNA without detectable AVP immunoreactivity. In the Brattleboro rat, staining intensity of the reaction was lower than in normal rat and the AVP mRNA was restricted mostly to the periphery of the cytoplasm. In this strain, the neurons containing the AVP mRNA did not contain AVP or oxytocin immunoreactivity. These results demonstrate that neuropeptide mRNA can be detected in semi-thin sections with a biotinylated oligonucleotide probe, and that AVP gene deletion provokes modification of the intracellular localization of the AVP mRNA.

Effect of colchicine on the immunohistochemical localization of somatostatin in the rat brain: light and electron microscopic studies.

Somatostatin (SRIF), the hypothalmic hormone which inhibits the secretion of growth hormone by the pituitary, has been localized immunohistochemically in the rat hypothalamus after intracerebral injection of colchicine. The number of cell bodies staining for SRIF was increased in the periventricular nucleus while the number of nerve fibers was decreased in the median eminence after treatment. The number of secretory granules containing SRIF in the nerve cell bodies was increased in the treated animals, suggesting a correlation between the number of specific secretory granules and intensity of the immunohistochemical reaction. These observations are in agreement with the hypothesis that SRIF cell bodies in the periventricular nucleus send their axons into the median eminence.

Anatomical studies of cholecystokinin in neurons and pathways involved in neuroendocrine regulation.

A CCK-like substance has been identified in two distinct neuronal systems of the PVN which are both involved in neuroendocrine regulation. The CCK parvicellular neurons are potentially involved in the regulation of adenohypophyseal hormone secretion through projections to the external zone of the median eminence. CCK immunoreactivity was also identified in the magnocellular-neurohypophyseal system, exclusively in oxytocinergic neurons. Thus, CCK might participate in the modulation of oxytocin secretion.

Immunohistochemical localization of corticotropin releasing factor (CRF) in the hypothalamus of the squirrel monkey, Saimiri sciureus.

Corticotropin releasing factor immunoreactive (CRF-IR) neuronal cell bodies and fibers have been localized in both the paraventricular and supraoptic nuclei of the hypothalamus of the squirrel monkey. The major projection from these nuclei is to the median eminence and neural stem. A few CRF-IR fibers were found in the dorsal pars nervosa primarily adjacent to the pars intermedia. A rostral projection of CRF-IR fibers is associated with the suprachiasmatic nucleus and continues to septal areas. A caudally projecting bundle of fibers was observed entering the midbrain in neuropil adjacent to the aqueduct. The location of CRF-IR components is also compared with those containing vasopressin (AVP).

Adrenocorticotropic hormone (ACTH) in pituitary colloid and extrapituitary tissues.

The level of ACTH in bovine pituitary intraglandular colloid (IGC/ACTH), the holocrine secretion of the marginal half of the intermediate lobe (IL) cells, was found to correlate with the concentration of this peptide in blood plasma (BP/ACTH), cerebrospinal fluid (CSF/ACTH), the paraventricular nuclei (PVN/ACTH) and the arcuate nuclei (AN/ACTH). The ACTH content in all sites was measured by radioimmunoassay (RIA). Although the IL is virtually avascular, the intraglandular lumen housing the IGC communicates nonetheless with the venous cavernous sinuses as well as the subarachnoid cerebral spinal fluid (CSF). Therefore, we content that the IGC serves as a transport medium whereby IL materials gain access to these extrapituitary sites.

Coexistence of CRF peptide and oxytocin mRNA in the paraventricular nucleus.

Several studies have reported coexistences of peptides in parvocellular neurons of the paraventricular nucleus (PVN). However, the coexistence of peptides in the magnocellular PVN is less clear. Controversy exists in particular about the coexistence of corticotropin-releasing factor (CRF) and oxytocin (OX). Although these peptides are present in distinct areas of the PVN, some overlap may exist. This study investigated a potential coexistence of OX and CRF in magno- and parvocellular PVN. The data demonstrate with clarity that neurons containing both the mRNA for OX and the peptide CRF are present in subpopulations of magnocellular and parvocellular neurons of the PVN.

Angiotensin II immunoreactivity in push-pull perfusates from the paraventricular nucleus.

Push-pull perfusion of the hypothalamic paraventricular nucleus in sodium pentobarbital anesthetized Sprague-Dawley rats indicates the release of angiotensin II-immunoreactive material in this area. Attempts to demonstrate a neuronal origin of this material by chemical depolarization with perfusate containing either 40 or 120 mM K+ were unsuccessful. However, this material does appear to be of central origin since intravenous infusion of arginine-vasopressin, a similar sized peptide, did not result in the appearance of increased levels of this substrate in the perfusate, indicating that the integrity of the blood-brain barrier was not compromised by the perfusion.