Distribution of alpha-, beta- and gamma-endorphins in the forebrain and diencephalon of the rat brain (immunohistochemical study)

The investigation performed by means of specific rabbit antisera is one of the stages for mapping peptides. This is necessary for revealing functional role of the endorphins in the CNS. The indirect method of Coons is applied in parallel series of frontal paraffin slices of the brain 10 mcm thick. Neurons containing alpha-, beta- and gamma-endorphins are localized in the same brain areas. These are structures of the palaeocortex (the prepiriform cortex and the diagonal area) and those of the hypothalamus (the supraoptic, arcuate, ventromedial, mammillary nuclei, anterior and posterior fields). Endorphinergic neural fibers run within composition of various conducting cerebral systems, such as the corpus collosum, fornix, internal and external capsules.     

Distribution of oxytocic and vasopressor activity in the rat diencephalon

The oxytocic and vasopressor activity was studied in five 1 mm thick, horizontal sections of the rat diencephalon. The diencephalon was cut frozen in dry ice. The sections obtained from identical parts of the diencephalon of 10 rats were homogenized together in 0.9% NaCl solution acidified with glacial acetic acid. The homogenate was heated to 100 degrees C for 5 min and centrifuged. The oxytocic activity of extracts was determined in vitro by, the method of Holton using the rat myometrium. The vasopressor activity was determined in vivo recording blood pressure in the carotid artery of rat by the method of Dekańaski. Oxytocic activity was found in all five sections of diencephalon and vasopressor activity in only two sections. The first section included the median eminence and ventral hypothalamus together with the supraoptic nucleus, the second section included the the dorsal hypothalamus with paraventricular nucleus, the third section--the ventral thalamus, the fourth section--the middle part of thalamus, the fifth section--the dorsal thalamus.     

Ontogeny of IgE-bearing lymphocytes in the rat

IgE-bearing lymphocytes were detected by immunofluorescence in the spleen of neonatal Hooded Lister strain rats within 24 hr after birth. The same cells were detected in the bone marrow as early as the 4th day after birth. Both fetal spleen and liver obtained 1 day before birth contained IgM-bearing cells but no detectable IgE-bearing cells. The proportion of IgE-bearing cells in the spleen and bone marrow increased during the neonatal period and reached an adult level within 3 to 4 weeks after birth. In adult Hooded Lister rats, IgE-bearing cells were 3 to 6% of total spleen cells and 1.5 to 2.2% of bone marrow cells. Most of the IgE-bearing cells from bone marrow cells. Most of the IgE-bearing cells from both newborn and adult animals carried IgM determinants on their surface. Capping experiments showed that epsilon chain determinants and mu chain determinants belonged to separate molecules. IgG2a-bearing lymphocytes were detected in the neonatal spleen as early as the 4th day after birth, but a significant number of these cells was not detected in the bone marrow until the 4th week. In newborn spleen the percentage of IgE-IgM double bearing cells was higher than that of IgG2a-bearing cells.     

Natriuretic peptide receptors in cultured rat diencephalon

To characterize the type of cell expressing natriuretic peptide receptors in the brain and the nature of these receptors, we conducted studies in primary cultured glial and neuronal cells derived from fetal rat diencephalon. The glial predominant cultures (95% of total cells and glial fibrillary acidic protein positive) expressed nearly a 10-fold greater specific binding of the natriuretic peptides to cell surface receptors compared with the neuron-predominant cultures. Scatchard analysis of binding studies with 125I-atrial natriuretic peptide (ANP) and 125I-brain natriuretic peptide (BNP) revealed a single class of receptors with dissimilar affinities (0.25 +/- 0.09 and 0.74 +/- 0.07 nM, respectively, n = 3 experiments p less than 0.01) but similar numbers of binding sites for both peptides (93 and 88 fmol/mg of protein, respectively). Cross-linking of 125I-ANP and BNP to cultured glia followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography identified distinct bands at either approximate Mr 130,000, or 102,000 and 66,000, corresponding to two high molecular weight (B) receptors and one low molecular weight (C) receptor described in other tissues. Different subtypes of astrocytes appeared to express different B receptors. Binding and cross-linking of radiolabeled ANP or BNP were competitively inhibited equally by unlabeled ANP or BNP, indicating that ANP and BNP probably bind the same receptors. The glial cultures functionally expressed a receptor(s) with guanylate cyclase activity; BNP was less potent than ANP in stimulating cGMP at lower concentrations. These results indicate that both high and low molecular weight natriuretic peptide receptors are expressed in astrocyte-predominant cultures from the fetal diencephalon and suggest that glia participate in several actions of ANP which are probably mediated through this area of the brain.     

Ontogeny of ECL cells in the rat.

ECL cells produce histamine and chromogranin A, and are restricted to the oxyntic mucosa of the stomach. ECL cell ontogeny has been studied in some detail in the rat. Using histidine decarboxylase immunostaining, the first ECL cells can be demonstrated at embryonic day 17. Immunoreactive histamine and chromogranin A appear one day later. At embryonic day 20, the vesicular monoamine transporter type 2 is also present in the ECL cells. Neonatally the ECL cell proliferation is slow; however, one to three weeks postnatally there is a rapid growth of ECL cells to populate the basal half of the glands. Gastrin is known to be an important stimulator of ECL cell activity and growth in the adult rat. As revealed in recent mouse gene knock out models gastrin does not seem to play a role in the early ECL cell differentiation and development.     

Electron-microscopic characterization of adrenergic axon terminals in the diencephalon of the rat

Summary The fine structure of adrenergic axon terminals was examined in the paraventricular nucleus of the thalamus (PNT) and in the hypothalamic arcuate nucleus-median eminence (ARC-ME) complex by use of phenylethanolamine-N-methyl transferase (PNMT) immunocytochemistry. In the PNT, immunoreactive terminals formed a dense and well-circumscribed plexus. In the ARC, labeled varicosities were less numerous and more evenly distributed. In the ME, they were scarce and confined to the inner zone. In all these areas, the diameter of immunoreactive varicosities ranged between 0.2 and 1.3 m; in the ME and in the transitional zone between the ARC and the ME, a population of larger boutons (>2 m) was also visible. All immunoreactive varicosities exhibited densely packed small, clear vesicles associated with a few large granular vesicles. In the PNT and the ARC, but not in the ME, they formed synaptic contacts with dendritic elements and were occasionally apposed to neuronal cell bodies. These axo-somatic appositions showed no junctional specializations. In the ME and transitional zone, immunoreactive terminals were frequently juxtaposed to, and occasionally established differentiated synaptic contacts with, tanycytes. These data support a transmitter role for adrenaline in the diencephalon and suggest that adrenaline plays a role in hypothalamo-hypophysiotropic regulation through interactions with neural and glial elements.     

Ontogeny of insulin receptors in the rat hemochorial placenta

Binding of 125I-insulin to rat placental membranes was time and protein concentration dependent, reversible, and specific. Unlabeled porcine insulin competed for 125I-insulin binding with an IC50 of 65 nM, while IGF-I was much less potent with an IC50 of 2.12 mM. Specific binding of 125I-insulin decreased during the second half of gestation from Days 11 to 19. Scatchard analysis of the binding data for membranes prepared from Gestation Days 11 and 19 yielded typical curvilinear plots which showed a marked decrease in the number of binding sites in late gestation placenta. Beginning on Day 14, insulin binding was characterized with isolated labyrinth and basal zone portions of the hemochorial placenta. There was no evidence for differences in Kd values or the number of binding sites in these two functionally distinct portions of the rat placenta. Crosslinking of 125I-insulin followed by SDS-PAGE showed a single protein with a molecular weight of 130,000 from placental tissues on Gestation Days 11 and 19 and confirmed a gestational decrease in the number of insulin receptors. In solubilized, lectin-purified preparations from placenta and liver membranes, insulin stimulated the phosphorylation of a Mr 95,000 protein. 32P-incorporation into this 95,000 protein was stimulated fivefold by insulin in Day 11 placenta receptor, whereas no detectable 32P-incorporation was found in Day 19 placenta. Thus, while the alpha- and beta-subunits of insulin receptors in mid and late gestation placenta have molecular weights which are similar to receptors in maternal liver, data indicate the presence of a functional difference in insulin-stimulated kinase activities.     

Ontogeny of histamine-immunoreactive cells in rat stomach

Summary An antiserum against hemocyanin-conjugated histamine was used to study the cellular stores of histamine in the stomach, especially the oxyntic mucosa, of fetal and early postnatal rats. Tissues were fixed in 4% 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC-DI) and standard immunofluorescence technique was used. Histamine was first detected on the 16th embryonic (E16) day when a few histamine-immunoreactive (HA-ir) cells and nerve fibers were observed in the muscular layer of the stomach wall. On day E18, HA-ir cells were visualized for the first time in the oxyntic mucosa of the stomach, and from that day on the number of such cells increased slowly initially and after day E20 more rapidly. At birth many of the HA-ir cells in the oxyntic mucosa possessed processes giving them a paracrine-like appearance typical of enterochromaffin-like cells (ECL cells). Only a very small number of the HA-ir cells represented metachromatically stained mast cells and were located in the submucosa. After birth, the number of HA-ir ECL cells increased steadily, until day 21 when the distribution and number was very similar to that of the adult. The results suggest that histamine-containing neurons and ECL cells appear in the stomach wall before birth, and that there are histamine-containing ECL cells in the mucosa and mast cells in the submucosa of the stomach wall at birth.     

Ontogeny and distribution of cysteine sulfinate transaminase in the rat brain

Cysteine sulfinate transaminase catalyzes the conversion of cysteine sulfinate and -ketoglutarate to -sulfonyl pyruvate and glutamate and is present in high concentration in neuronal tissue. During development, levels of cysteine sulfinate transaminase in whole rat brain homogenate increase twofold between days 10 and 21. At maturity, high levels of the enzyme are present in the synaptosomal fraction. The highest level of enzyme activity was in the hypothalamus, approximately three- to fourfold greater than any other region examined. These data suggest that cysteine sulfinate transaminase may have an important role in the metabolism of neurotransmitters and their precursors.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

Summary In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (CIR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (C-IR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Ontogeny of absence-like and tonic seizures in the spontaneously epileptic rat

The ontogeny of epileptic seizures in spontaneously epileptic rats (SER; zi/zi, tm/tm) was studied by examining behaviour and electroencephalogram (EEG) simultaneously. Weight gain and survival time were also studied. Compared with the control Kyo:Wistar rats, SER showed a much smaller increase in body weight. All male and female SER died before 20 and 18 weeks of age, respectively. Body tremor was observed at 2 weeks of age but disappeared after 11 weeks. Staggering gait appeared after 7 weeks of age, and intensified with age. Absence-like seizures characterized by paroxysmal appearance of 5-7 Hz spike-wave-like complexes were observed in the cortical or hippocampal EEG after 5 weeks of age, and tonic seizures with low voltage fast waves were observed after 6 weeks of age. All SER exhibited both absence-like and tonic seizures with high frequencies from 12 weeks of age. Differences with other spontaneous rat models of epilepsy and application methods for estimating seizure-inhibitory effects of anti-epileptic drugs are discussed.     

Ethanol alters microvasculature enzymes in the rat forebrain

Forebrain arterioles were analyzed histochemically to determine the effects of an acute administration of ethanol on key enzymes of aerobic and anaerobic metabolism as well as on the hexose monophosphate shunt in rats. The enzymes were glucose 6-phosphate dehydrogenase, cytochrome oxidase, lactate dehydrogenase, beta-hydroxybutyrate dehydrogenase, and isocitrate dehydrogenase. All enzymes were quantified under two conditions: 1 h and 2 days after ethanol administration. Significant changes were noted in four of the five enzymes measured after 1 h and in all five enzymes when measured 2 days after ethanol administration. Our data suggest that ethanol may cause impaired metabolism in the forebrain microvasculature, which, in turn, may account for some of the characteristic behavioral effects of acute ethanol administration.     

Cytosolic sialidase in developing rat forebrain

The nature and developmental profile of the soluble sialidase of rat forebrain were studied from birth to 150 days. Forebrain was extracted by two procedures, one (mild) preserving, the other (drastic) destroying nerve endings. The soluble extracts obtained by the mild procedure contained 64–78% of the total tissue cytosol, assayed as lactate-dehydrogenase; those obtained by the drastic procedure 87–94%. These latter extracts were considered as the soluble fraction containing ‘all’ tissue cytosol. The cytosolic origin of the sialidase contained in the soluble extracts at all examined ages was suggested by the following evidence: (a) during extraction sialidase behaved as lactate-dehydrogenase and quite differently from β-hexosaminidase and β-galactosidase, enzymes of lysosomal nature present in the same extracts, (b) the sialidase content of the extract was not influenced by the presence or absence of EDTA in the medium, (c) the sialidase content in the extracts did not diminish even after prolonged centrifugation (2 h) at high speed (150,000 g). The content of cytosolic sialidase referred to g fresh tissue increased from birth to 20 days, and slowly decreased thereafter. Till 20 days the content and the developmental trend of the cytosolic enzyme were similar to that of the better known membrane bound sialidase. This latter enzyme, however, reached its maximum at about 60 days of age. The specific activity of the cytosolic sialidase was lower till 10 days of age, higher from 10 to 30 days, and equalled that of the membrane bound enzyme during adult life. Therefore rat forebrain cytosolic and membrane bound sialidases, also from the developmental point of view, behave as different enzymes.     

Ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland

Summary The ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland has been studied using high iron diamine (HID), Alcian Blue (AB) at pH 1.0, high iron diamine in combination with Alcian Blue at pH 2.5 (HID-AB), cationic colloidal gold (CCG) at pH 1.0 under light microscopy and CCG (1.0), HID-thiocarbohydrazide (TCH)-silver proteinate (SP)-physical development (PD) under electron microscopy. From day 19.5 of gestation, sulphated glycoconjugate-producing cells were discernible under both light and electron microscopy. The development of such cells can be classified into four stages: (1) a prenatal period from day 19.5 of gestation extending to 0.5 days after birth; (2) 1 day to 2 weeks after birth; (3) 2 to 4 weeks after birth; and (4) the final period from 4 to 8 weeks after birth. Glycoconjugate-producing cells reached maturity by 4 weeks after birth. Our results indicated that glycoconjugate-producing cells were cells along the wall of foveolar lumen, but not those covering the gastric mucosa surface. Our results also suggested that thetrans totransmost Golgi apparatus lamellae were the sites of sulphation in the developing rat stomach.     

Ontogeny and implication of pregnancy-associated agglutinin of the rat uterus

The mannose/fructose-binding agglutinin from Day 1-7 post coitum (p.c.) rat uteri was purified on Concanavalin A. The specific haemagglutination activity peaked on Days 4 and 5 p.c. and a 1.4-fold increase in the yield was accompanied by a 10-12-fold increase in specific agglutination titre. The mannose-binding affinity of the protein also increased, but the highest fructose-binding affinity was found on Day 1 p.c., which may indicate a role of the protein in fructose concentration for utilization by the spermatozoa. Rats that were pseudopregnant, superovulated and pseudopregnant, and had one uterine horn ligated showed that, although a basal level of the protein was induced by the hormonal milieu, actual stimulation of the protein synthesis occurred in the presence of the fertilized ova.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of A- and B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to B-crystallin, but not to A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to A- and B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to A- and B-crystallin. In rat embryos, A-crystallin appeared in the lens pit at E12, and B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to A-crystallin. The lens fiber cells were also immunoreactive to B-crystallin, but the epithelial cells were not. These findings suggest that B-crystallin appears earlier than A-crystallin in the human lens, but at a later period than A-crystallin in the rat lens. B-Crystallin was not detected in the epithelial cells of the rat lens, but was perisistently present in the epithelial cells of the human lens.