Development changes in long-form leptin receptor expression and localization in rat brain.

The expression and localization of long-form leptin receptor (OB-Rb) were studied immunocytochemically in the brain of fetal and adult rats using a polyclonal antibody that specifically recognized OB-Rb. At 14 days of gestation, immunoreactive cells were observed in the ventricular layer, which contains premature neuronal cells. At 18 days of gestation, they were weakly stained but obvious in the paraventricular nucleus (PVN), and ependymal cells also showed immunoreactivity. At birth, the immunoreactivity of OB-Rb in the PVN seemed to be much lower than that in adult rats and remained low during the suckling period. Considering the presence of neuroendocrine and structural neuronal abnormalities in Lepob/Lepob mice with genetic leptin deficiency, these results suggest that the expression of OB-Rb in premature neuronal cells may have some function, and that the regulation of energy balance by leptin through hypothalamic regions, such as PVN, may not yet be developed in the perinatal period.

Hypophysiotrophic TRH-producing neurons identified by combining immunohistochemistry for pro-TRH and retrograde tracing.

To determine hypophysiotrophic thyrotropin-releasing hormone (TRH)-producing neurons in the rat hypothalamus, we employed a combination of the immunohistochemistry for TRH prohormone (pro-TRH) and the retrograde tracing of neurons that project to the median eminence (ME) by injecting biotinylated wheat germ agglutinin (WGA) into the ME. In intact rats, immunoreactive pro-TRH-positive neurons occurred in the parvicellular paraventricular nucleus (parvi-PVN), basal part of the anterior and lateral hypothalamus, perifornical area and dorsomedial nucleus, especially accumulating in the parvi-PVN. Twenty-four hours after injection of the WGA into the middle portion of the ME, we found neurons that incorporated the lectin in the anterior periventricular area, the PVN, and the arcuate nucleus. When we examined serial sections consecutively stained with anti-WGA, anti-pro-TRH, and anti-WGA, most of the pro-TRH-labeled neurons in the medial parvi-PVN and a part of the neurons in the anterior periventricular area and in the anterior, lateral, and dorsal parvi-PVN appeared to incorporate WGA. These neurons may correspond with the hypophysiotrophic TRH-synthesizing neurons in the rat hypothalamus.

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein in tanycytes of the mediobasal hypothalamus: distribution and relation to dopamine and luteinizing hormone-releasing hormone neurons and other glial elements.

The distribution of a dopamine- and cyclic adenosine-3':5'-monophosphate (cAMP)-regulated phosphoprotein with an apparent molecular weight of 32,000 (DARPP-32) was investigated in the rat diencephalon and monkey hypothalamus by use of immunohistochemical techniques. In addition to single cells located peri- and paraventricularly in hypothalamus and thalamus in the rat, and ependymal cells, DARPP-32-immunoreactivity was found to be present in a subpopulation of ependymal tanycytes. These DARPP-32-positive tanycytes lined the walls and floor of the third ventricle, sending processes towards the arcuate nucleus, surrounding blood vessels in this nucleus, and continuing towards the median eminence, where they abutted on portal vessels. A second group of DARPP-32-positive tanycytes with cell bodies within the median eminence was also observed. Simultaneous labeling with antiserum against tyrosine hydroxylase, a presumptive marker for tuberoinfundibular dopamine neurons, revealed a close relation to DARPP-32-containing tanycytes in several anatomical locations. Thus, in the periventricular area DARPP-32-positive tanycytes ensheathed tyrosine hydroxylase-positive processes. These processes, presumably representing dopaminergic dendrites, virtually penetrated between the ependymal cells to the ventricular space and thus perhaps established direct contact with the cerebrospinal fluid. Tyrosine hydroxylase-terminals were also observed in close association with DARPP-32-immunoreactive tanycytes in the rat median eminence. However, in view of the density of DARPP-32-positive processes in the external layer of the median eminence, the DARPP-32 processes may be related to a number of other types of nerve endings, including luteinizing hormone-releasing hormone, as shown in this study. The close association of DARPP-32-immunoreactive processes with tyrosine hydroxylase- and luteinizing hormone-releasing hormone-immunoreactive nerve endings in the rat was directly visualized at the ultrastructural level using triple-labeling immunocytochemistry. Both the ultrastructural analysis and immunohistochemistry at the light microscopic level, comparing the distribution of DARPP-32 and glial fibrillary acidic protein, indicated the presence of two types of glial processes in the median eminence. The electron microscopic studies also suggested the presence of both DARPP-32-positive and DARPP-32-negative glial processes in the external layer of the median eminence.(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)

Differential immunolabeling for electron microscopy of diverse peptidergic neurons.

We describe a simple and reliable method for differential immunolabeling of pre- and post-synaptic signal peptides at the ultrastructural level. Hypothalamic tissues of rats, including the suprachiasmatic nucleus, were cut on a Vibratome. Visualization of the immunolabeling of somatostatin (SRIH) and vasoactive intestinal polypeptide (VIP) was performed with avidin-biotin-peroxidase-diaminobenzidine (DAB). The end product of the DAB to VIP was further silver-intensified in a physical processing using silver nitrate, and the silver grains were finally substituted for gold. DAB-labeled SRIH fibers synapse on gold-labeled VIP perikarya and dendrites in the suprachiasmatic nucleus.

Coexistence of TRH with other neuroactive substances in the rat central nervous system.

Colocalization of thyrotropin-releasing hormone-like immunoreactivity with other neuroactive substances was examined immunohistochemically in colchicine-treated rat brains using double-staining or elution-restaining methods. Thyrotropin-releasing hormone-like immunoreactivity was shown to be located in the same neurons as: 1. enkephalin-, gamma-amino butyric acid- and tyrosine hydroxylase-, but not somatostatin-like immunoreactivity in the glomerular layer of the olfactory bulb 2. oxytocin- and cholecystokinin-, but not vasopressin-like immunoreactivity in the supraoptic nucleus 3. cholecystokinin-like immunoreactivity in posterior pituitary 4. enkephalin-like immunoreactivity in the perifornical area of the hypothalamus and 5. neuropeptide Y- and neurotensin-like immunoreactivity in the periaqueductal central grey. These findings provide further examples of coexistence of thyrotropin-releasing hormone with classical neurotransmitters and/or peptides in the rat central nervous system.

Distribution of TRH-like immunoreactivity with special reference to coexistence with other neuroactive compounds.

During the last years, several important advancements have been made that are of importance for our understanding of the distribution and localization of neurons and cells producing TRH-LI. As detailed in other chapters in this volume, the precursor for TRH has been characterized that has allowed production of antibodies raised against specific sequences of this precursor. This, in turn, has provided new tools for the immunohistochemical elucidation of TRH systems in the CNS. The TRH precursor has also been cloned, leading to possibilities for studying the localization of TRH mRNA with in situ hybridization. Finally, as shown in this paper, improvement of the fixation technique has made it possible to visualize extensive TRH-immunoreactive cell body and fiber systems with antiserum raised against the TRH tripeptide. The results from the latter studies and those with antisera directed to the TRH precursor and in situ hybridization are in good agreement, with some minor exceptions. It should be pointed out that some of the systems described here, for example TRH positive-cell bodies in cortical areas and the hippocampal formation, contain only a very weak immunoreactivity. As always with immunohistochemical techniques, the possibility of crossreactivity with TRH-like peptides or TRH-like sequences within larger proteins must be considered. The present results confirm the presence of TRH-LI in the insulin-producing beta cells of the pancreas, which with the improved technique can be demonstrated also in early adulthood in rats and guinea pigs. Moreover, it could be established that TRH-LI is present in neurons in the gastrointestinal tract as well as in a population of endocrine cells in the antrum of the stomach of the guinea pig. These cells seem at least partly to be identical to the well-known gastrin-producing cells. TRH-LI has been observed to occur in neurons already containing a classical transmitter and/or other peptides. Of particular importance here seems to be a descending bulbospinal system that in addition to TRH co-contains 5-HT, substance P-LI, galanin-LI, human growth hormone immunoreactive material, and proctolin-like material. The significance of this coexistence is not well understood, but interesting interactions have been observed. Attempts to manipulate the TRH phenotype in these medullary neurons by transplantation to other sites in the brain has so far shown that the expression of this peptide seems fairly stable.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of serum-free culture conditions on subcellular localization of steroid 5alpha-reductase in rat C6 glioma cells.

Rat C6 glioma cells are considered to be well characterized, and therefore commonly used as a model system to investigate the function of glial cells. However, recent study has shown that an alteration in the expression of their phenotypic antigens is observed when the cells are maintained under the serum-free conditions, proposing the possibility that various properties of glioma cells can be altered by the growth conditions. To test this possibility, the effects of serum-free culture conditions on the expression of steroid 5alpha-reductase (5alpha-R) type 1 isozyme in glioma cells were examined using immunocytochemical technique. Immunoreactivity of 5alpha-R type 1 was confined to the perinuclear region of glioma cells cultured in serum-containing medium, and observed in the cytoplasmic space as well as the perinuclear region of the cells cultured in serum-free medium. In contrast, serum deprivation failed to affect the expression of phenotypic antigens, glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Further studies showed that the expression of cytoplasmic 5alpha-R immunoreactivity induced by serum deprivation was reversible, and might be attributed to removal of serum proteins rather than biologically active small molecules from culture medium. This alteration in the expression of 5alpha-R immunoreactivity is therefore considered to reflect the translocation of the enzyme from the perinuclear region to the cell cytoplasm rather than the induction of cytoplasmic enzyme, and suggest that the culture conditions cause an alteration in the subcellular localization of 5alpha-R type 1 isozyme without phenotypic change of the glioma cell.

Thyrotropin-releasing hormone (TRH)-immunoreactive neuron populations in the rat olfactory bulb.

Thyrotropin-releasing hormone (TRH)-like immunoreactivity was examined in the rat olfactory bulb by an immunohistochemical method using antiserum against the tripeptide TRH. TRH-immunoreactive neurons and neuronal processes were localized in both main and accessory olfactory bulb, including the glomerular layer, external plexiform layer, mitral cell body layer, internal plexiform layer and granule cell layer. However, no TRH-positive structures were observed in the olfactory nerve layer. The present study demonstrates that TRH is located in several neuronal populations in the olfactory bulb, and that this peptide thus may be involved in the processing of information in the olfactory system.

Immunocytochemical localization of lung-type prostaglandin F synthase in the rat spinal cord.

Prostaglandin F synthase, producing prostaglandin F(2 alpha) and 9 alpha,11 beta-prostaglandin F(2), has at least two isozymes, lung-type and liver-type ones. The present study including double immunolabelling with microtubule-associated protein 2 indicated that the lung-type isozyme was present in neuronal dendrites and somata of gray matter (relatively intense in lamina I and II in dorsal horn, and IX in ventral horn) and vascular endothelial cells in the rat spinal cord at all segmental levels.

Substance P-like immunoreactive neurons in the tuberoinfundibular area of rat hypothalamus. Light and electron microscopy.

Immunoreactive substance P (ir SP) neurons were examined with special reference to their links with other neurons and blood vessels in the tuberoinfundibular hypothalamic area of normal and colchicine-treated male rats. On intraventricular administration of colchicine, ir SP neuronal cell bodies appeared in both the arcuate nucleus and the ventral portion of the ventromedial nucleus. These ir fibers were seen in the nuclei of untreated animals closely surrounding ir methionine-enkephalin and ACTH cell bodies, suggesting the presence of synaptic junctions. Electron microscopic examination of the arcuate nucleus stained with anti-SP serum showed synapses of ir SP fiber terminals on unlabeled neuronal cell bodies and fibers, some of which might be methionine-enkephalin and ACTH neurons. The ir fiber terminals contained numerous small clear and/or cored vesicles and a few large cored vesicles. Fiber terminals with the same ultrastructural characteristics were seen protruding into the pericapillary space in the external and subependymal layers of the median eminence. The possible role of the SP neurons in the tuberoinfundibular area is discussed.

Hypothalamic substance P-containing neurons. Sex-dependent topographical differences and ultrastructural transformations associated with stages of the estrous cycle.

Immunoreactive substance P (ir SP)-containing neurons were examined in rat hypothalamus. In untreated males, few if any ir perikarya were found in the arcuate nucleus, but after intraventricular colchicine administration, many appeared not only in the arcuate nucleus but also in the premammillary region, posterior hypothalamic, ventromedial, dorsomedial, subthalamic and paraventricular nuclei, and zona incerta. This was also the case in females treated with colchicine on the second day of diestrus. In untreated females, perikarya were seen only in the arcuate nucleus, varying in number and ultrastructure during the estrous cycle and being maximal in number in proestrus and estrus. The perikarya possessed well-developed Golgi bodies and lamellar bodies composed of many closely apposed cisternae of endoplasmic reticulum in proestrus, and showed stacks of two or three cisternae of endoplasmic reticulum as well as lysosomes in estrus. In diestrus, the perikarya had only a few granulated vesicles and fragmental cisternae of endoplasmic reticulum. In the perikarya of colchicine-treated males, many granulated vesicles and lysosomal bodies were noted. These findings suggest that, among the widely distributed SP neurons in the hypothalamus, some in the arcuate nucleus are involved in the hypothalamic-hypophysial-gonadal axis.

Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors.

Glucocorticoids have been shown to be neurotoxic and appear to play a role in neuronal cell loss during aging and following neuropathological insults. However, very little is known about the effects of these steroid hormones on glial cells. The effect of the synthetic glucocorticoid dexamethasone (DEX) on glial cell viability was therefore examined by measuring neutral red uptake into rat C6 glioma cells. Serum deprivation markedly reduced cell viability, and this effect was significantly enhanced by DEX. Electrophoretic analysis showed that the cell damage induced by either serum deprivation alone or in combination with DEX was not accompanied by the degradation of DNA into nucleosomic fragments. Electron microscopic studies confirmed that serum deprivation and glucocorticoid treatment caused necrotic cell death. Furthermore, the effect of DEX on cell viability could be mimicked by the glucocorticoid receptor agonist RU28362, and completely prevented by the glucocorticoid receptor antagonist RU38486. These results indicate that dexamethasone can enhance the necrotic death of glioma cells induced by serum deprivation, suggesting that glucocorticoids may be involved in the chronic alteration of brain function arising from neuropathological damage to glial cells.

Immunohistochemical evidence for synaptic connections between neuropeptide Y-containing axons and periventricular somatostatin neurons in the anterior hypothalamus in rats.

By employing a pre-embedding double immunolabeling technique, we examined light and electron microscopically synaptic associations between neuropeptide Y (NPY)-containing axons and somatostatin (SRIH)-containing neurons in the anterior periventricular area (APV) of the rat hypothalamus. For light microscopy, the immunoreactions for NPY and SRIH were visualized with silver-gold and diaminobenzidine (DAB), respectively, and the reverse labeling was used for electron microscopy. Light microscopy disclosed many brown SRIH perikarya surrounded by several black beads of NPY fibers in the APV. In electron microscopy, immunoreactive SRIH neurons revealed silver-gold particles scattered throughout the cytoplasm and accumulated in the Golgi area and the secretory granules. SRIH perikarya and dendritic processes indicated synaptic associations with DAB-labeled NPY fiber terminals and immunonegative fibers. NPY presynaptic terminals possessed numerous small clear vesicles and a few dense core vesicles; vesicular membranes and cores were labeled with DAB chromogen. Both the pre- and postsynaptic membranes were thickened equally to be a symmetric synapse. These findings suggest that NPY neurons are involved in the regulation of growth hormone secretion from the pituitary by affecting periventricular SRIH neurons.

Immunocytochemical evidence for synaptic regulation of paraventricular vasopressin-containing neurons by substance P.

Employing a combination of pre-embedding peroxidase-antiperoxidase-labeling for substance P (SP) and postembedding immunogold labeling with protein A-colloidal gold-anti-arginine vasopressin (AVP) complex, we demonstrated immunoreactive SP containing nerve fibers, which terminate synaptically on the perikarya, contained gold-labeled secretory granules in the magnocellular paraventricular nucleus of rats. The perikarya were also synapsed with unlabeled nerve fibers. It is concluded that SP plays a role as an axosomatic neurotransmitter in diverse synaptic controls of vasopressinergic neurons.

Immunohistochemical presence of 5 alpha-reductase rat type 1-containing cells in the rat brain.

We showed immunohistochemically the localization of 5 alpha-reductase-containing cells in the rat brain, using a rabbit antibody generated against 5 alpha-reductase rat type 1. The antibody was produced by injecting the synthetic peptide corresponding to the amino acids 38-53 of 5 alpha-reductase rat type 1, conjugated to keyhole limpet hemocyanin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Western blot analysis revealed that this antiserum recognized the protein with a molecular weight of 29,000 Da. The immunoreactive cells were distributed throughout the brain and they were preferentially located in the white matter rather than in the grey matter. These cells were mostly small and round and had a few fine processes. The immunoreaction was confined to the cytoplasm and processes. These findings indicate that 5 alpha-reductase rat type 1-containing cells are widely distributed in the rat brain and are located preferentially in the white matter rather than in the grey matter.

Influence of serum-free culture conditions on steroid 5alpha-reductase mRNA expression in rat C6 glioma cells.

Immunocytochemical studies previously showed that serum deprivation resulted in the appearance of steroid 5alpha-reductase (5alpha-R) in the cytoplasm of rat C6 glioma cells. To determine whether this increase in cytoplasmic 5alpha-R was due to changes in 5alpha-R gene expression, the effect of serum deprivation on 5alpha-R mRNA expression was examined. No significant change in the mRNA levels was observed in cells grown in serum-free culture medium. Therefore, the appearance of 5alpha-R immunoreactivity in the cell cytoplasm observed under serum-free conditions is probably not due to changes in 5alpha-R gene expression.

Hypothalamic thyrotropin-releasing hormone (TRH)-containing neurons involved in the hypothalamic-hypophysial-thyroid axis. Light microscopic immunohistochemistry.

The localization of neurons containing immunoreactive thyrotropin-releasing hormone (TRH) was examined in the hypothalamus of intact, propylthiouracil (PTU)-treated, and colchicine-treated adult rats. In intact animals, immunoreactive TRH neurons were occasionally found in the paraventricular and dorsomedial nuclei, and in the anterior and lateral hypothalamic areas. In PTU-treated animals, the cellular appearance of the hypothalamus with the exception of the paraventricular nucleus was almost similar to that of intact animals. In the paraventricular nucleus, only the cells localized in the periventricular and medial parvocellular subdivisions significantly increased in number and became hypertrophic in comparison with intact animals. The distribution of immunoreactive fibers in the hypothalamus was almost equal among the 3 animal groups with the exception of that in the median eminence, in which the fibers were most densely concentrated in intact animals, and most sparse in PTU-treated rats. The fibers projecting into the median eminence were distinguished into the periventricular and lateral pathways, which are derived from the neurons in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus, respectively. Thus, among immunoreactive TRH neurons in the hypothalamus, only those in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus may be involved in the hypothalamic-hypophysial-thyroid axis.

Development of immunoreactive lhrh neurons in the fetal rat hypothalamus.

The ontogenesis of immunoreactive (ir) LHRH neurons was investigated in rats applying the double-bridge PAP method of Vacca et al. (J. Histochem. Cytochem. 28, 297-307, 1980). Ir LHRH was first evident in the cell bodies confined in the ventromedial surface of the anterior part of the forebrain vesicles on day 16.5 of gestation. Only one or two cells, if any, were found in the brains examined; the cells are oval showing ir brown granules within the perikarya and also within the processes extended from both poles. On day 17.5 of gestation, a few ir cells appeared in the olfactory cortex, medial septum, medial preoptic area, diagonal band of Broca, and ventro-lateral surface of the anterior hypothalamus, and, with development, their numbers increased gradually. During the prenatal period, no ir cell exists in the arcuate nucleus or in the medial-basal hypothalamus. Beaded ir fibers appeared in the organum vasculosum of the lamina terminalis (OVLT) and in the external layer of the median eminence on 18.5 and 19.5 days of gestation, respectively. The present findings ascertain that the hypothalamic regulation of hypophysial gonadotrophic function exists during fetal period in rats.

Development of the neuronal system containing neuropeptide Y in the rat hypothalamus.

In the rat hypothalamus, neuropeptide Y-containing neurons first appeared on day 14.5 of gestation in the arcuate nucleus and in the dorsolateral hypothalamic area. Until birth neuropeptide Y-containing cell bodies increased in number in the arcuate, dorsomedial-lateral and paraventricular nuclei, but disappeared thereafter, but some cells remaining in the arcuate nucleus. In animals treated neonatally with monosodium L-glutamate to destroy the arcuate nucleus, neuropeptide Y-immunoreactivity became evident in many cells scattered in the magnocellular paraventricular and dorsomedial-lateral hypothalamic nuclei on day 16 but not on days 60 and 12. These neuropeptide Y-immunoreactive neurons which appeared in the paraventricular nucleus were also vasopressin-positive. Neuropeptide Y fibers, on the contrary, remarkably diminished in number on day 16, particularly in the paraventricular and dorsomedial-lateral nuclei, and the medial preoptic area, but made a considerable recovery on days 60 and 120. Hence it is probable that, in normal ontogenetic progress, the development of the neuropeptide Y fibers in these areas is inhibitorily affected by that of arcuate neuropeptide Y neurons.