scholarly journals ISOLATION OF NERVE ENDINGS FROM THE POSTERIOR PITUITARY GLAND

1965 ◽  
Vol 25 (3) ◽  
pp. 179-193 ◽  
Author(s):  
Frank S. Labella ◽  
Madhu Sanwal
Keyword(s):  
Electron Microscopic Examination ◽  
Basement Membranes ◽  
Nerve Endings ◽  
Posterior Pituitary ◽  
Neurosecretory Granules ◽  
Electron Microscopic ◽  
Free Nerve Endings ◽  
Pituitary Glands ◽  
Posterior Pituitary Gland ◽  
Staining Techniques

Bovine posterior pituitary glands were homogenized in 10 per cent sucrose and fractionated by differential centrifugation. The following centrifugation procedure resulted in the most satisfactory separation: 1000 g for 15 minutes—nuclei, connective tissue, basement membranes with associated endothelium, giant nerve endings, and whole pituicytes; 4200 g for 15 minutes—free nerve endings, including Herring bodies; 17,000 g for 15 minutes—mitochondria; 68,000 g for 15 minutes—neurosecretory granules. Electron microscopic examination was carried out on whole tissue and on the isolated fractions. Isolated nerve endings were examined also by negative staining techniques. Isolated nerve endings retain an apparently normal complement of mitochondria, neurosecretory granules, and microvesicles ("synaptic" vesicles). The free nerve endings closely resemble those observed in sections of intact posterior pituitary tissue. Free microvesicles were not observed in any of the fractions isolated and apparently sediment at centrifugal forces higher than those employed in this study.

scholarly journals Immunoelectron microscopic characterization of vasopressin neurons in macaques by use of formaldehyde-fixed tissues stored for several years

2021 ◽  
Author(s):  
Akito Otubo ◽  
Sho Maejima ◽  
Takumi Oti ◽  
Kaita Satoh ◽  
Yasumasa Ueda ◽  
...  
Keyword(s):  
Translational Research ◽  
Median Eminence ◽  
Glutamate Transporter ◽  
Microscopic Analysis ◽  
Nerve Endings ◽  
Size Difference ◽  
Posterior Pituitary ◽  
Electron Microscopic ◽  
Vasopressin Neurons

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed with 4% formaldehyde and stored at −25°C for several years. The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 were colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

scholarly journals Morphologic Characterization of Castor Oil-induced Colitis in Ponies

1993 ◽  
Vol 30 (3) ◽  
pp. 248-255 ◽  
Author(s):  
C. M. Johnson ◽  
J. M. Cullen ◽  
M. C. Roberts
Keyword(s):  
Lamina Propria ◽  
Castor Oil ◽  
Electron Microscopic Examination ◽  
Neutrophil Infiltration ◽  
Basement Membranes ◽  
Electron Microscopic ◽  
Cytoplasmic Matrix ◽  
Severe Diarrhea ◽  
Mucosal Erosion ◽  
Junctional Complexes

Ten ponies (160-250 kg, ages 17 months to 20 years) developed severe diarrhea within 24 hours of castor oil administration (2.5 ml/kg orally). The diarrhea was most severe between 24 and 48 hours post-dosing and subsided by 72 hours. Ponies were euthanatized at 24, 48, and 72 hours post-dosing and intestine was evaluated histologically and ultrastructurally. Twenty-four hours after dosing, the mucosa of the cecum and ventral colon had extensive superficial epithelial erosion and neutrophil infiltration. In the ileum, the epithelium of villous tips was separated from the lamina propria. Scanning electron microscopic examination of the cecal mucosa revealed that basement membranes were exposed in most areas except within necks of crypts. Ultrastructurally, changes in superficial enterocytes of the cecum and ventral colon were characterized by loss of microvilli, distortion of the cytoplasmic terminal web, expansion of the cytoplasmic matrix with formation of precipitates, and widening of intercellular spaces between junctional complexes. Enterocytes located within necks of crypts were flattened along the basement membrane and extended to margins of erosions. Venules within the superficial lamina propria were occluded by fibrin thrombi. Erosions in the cecum and ventral colon of ponies examined 48 hours after treatment were less extensive than those of ponies examined at 24 hours. At 48 hours post-dosing, basement membranes adjacent to crypts were covered by cuboidal enterocytes characterized ultrastructurally by sparse, irregularly shaped microvilli located on broad cytoplasmic protrusions and by numerous free ribosomes. These features indicated that immature enterocytes had migrated from crypts to resurface the eroded mucosa. By 72 hours post-dosing, the entire mucosa was covered by columnar epithelium, although cells were shorter and had decreased density of microvilli than cells of control animals. A single dose of castor oil consistently produced transient diarrhea associated with neutrophilic inflammation and mucosal erosion in the cecum and ventral colon. Resolution of diarrhea occurred shortly after epithelial restitution.

ISOLATION OF RADIOACTIVE OXYTOCIN AND VASOPRESSIN FROM THE POSTERIOR PITUITARY GLAND OF THE RAT AFTER THE INJECTION OF LABELLED TYROSINE INTO THE CEREBROSPINAL FLUID

1971 ◽  
Vol 49 (1) ◽  
pp. 93-103 ◽  
Author(s):  
B. T. PICKERING ◽  
C. W. JONES
Keyword(s):  
Cerebrospinal Fluid ◽  
Pituitary Gland ◽  
Posterior Pituitary ◽  
Intracisternal Injection ◽  
Pituitary Glands ◽  
Posterior Pituitary Gland ◽  
High Degree ◽  
Very High

SUMMARY A method is described for the preparation of isotopically pure [3H]oxytocin and [3H]vasopressin from the pooled posterior pituitary glands of groups of four or five rats after the intracisternal injection of [3H]tyrosine. Hormones were separated from deproteinized neurohypophysial extracts by chromatography on Amberlite CG-50, and further purified by chromatography on carboxymethylcellulose. In this way, samples of the hormones were obtained with a very high degree of isotopic purity. The method is suitable for studying the effects of stimuli to the hypothalamo—neurohypophysial system on the biosynthesis and transport of the hormones.

DISTRIBUTION OF CORTICOTROPHIN RELEASING FACTOR ACTIVITY WITHIN THE HYPOTHALAMIC-PITUITARY COMPLEX OF RATS AND CATTLE

1977 ◽  
Vol 75 (2) ◽  
pp. 293-303 ◽  
Author(s):  
NAOKI YASUDA ◽  
MONTE A. GREER ◽  
SUSAN E. GREER ◽  
PATRICIA PANTON
Keyword(s):  
Pituitary Gland ◽  
Dose Response ◽  
Median Eminence ◽  
Response Curve ◽  
Posterior Pituitary ◽  
Corticotrophin Releasing Factor ◽  
Pars Nervosa ◽  
Pituitary Glands ◽  
Posterior Pituitary Gland ◽  
Qualitative Similarity

An assay system involving cultured rat adenohypophysial cells from either intact or adrenalectomized donors was used to study the distribution of corticotrophin releasing factor (CRF) activity in the hypothalamic-pituitary complex of rats and cattle. In the rat hypothalamus, CRF activity was most concentrated in the median eminence, but CRF was present in the stalk and the posterior pituitary gland in much higher concentrations than in the median eminence in both species. The dose–response slopes for the median eminence, stalk and pars nervosa of the posterior pituitary gland were parallel to each other, suggesting a qualitative similarity between the CRF activity in these tissues. Rat posterior pituitary glands may also contain another CRF component which has a much flatter dose–response curve, but is detectable in smaller quantities of posterior pituitary tissue than is the other type of CRF.

scholarly journals Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at –25 °C for Several Years

2021 ◽  
Vol 22 (17) ◽  
pp. 9180
Author(s):  
Akito Otubo ◽  
Sho Maejima ◽  
Takumi Oti ◽  
Keita Satoh ◽  
Yasumasa Ueda ◽  
...  
Keyword(s):  
Translational Research ◽  
Median Eminence ◽  
Glutamate Transporter ◽  
Microscopic Analysis ◽  
Nerve Endings ◽  
Size Difference ◽  
Posterior Pituitary ◽  
Electron Microscopic ◽  
Post Mortem Brain

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at –25 °C for several years (4–6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

THE TENTATIVE IDENTIFICATION OF THREE NEUROPHYSINS FROM THE RAT POSTERIOR PITUITARY GLAND

1972 ◽  
Vol 55 (3) ◽  
pp. 577-589 ◽  
Author(s):  
W. B. WATKINS
Keyword(s):  
Biochemical Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Posterior Pituitary ◽  
Tentative Identification ◽  
Pituitary Glands ◽  
Starch Gel ◽  
Posterior Pituitary Gland ◽  
Neurophysin Ii ◽  
Posterior Pituitary Lobe

SUMMARY Acetone-dried rat posterior pituitary glands were extracted with 0·1mhydrochloric acid and the soluble proteins fractionated by Sephadex gel exclusion and ion exchange chromatography. Three proteins had chromatographic and biochemical properties expected of neurophysins and also cross-reacted immunologically (as demonstrated by microimmuno-diffusion and -electrophoresis in agarose) with an anti-serum raised against porcine neurophysin-II. The three proteins were named neurophysin-I, -II and -III in order of their electrophoretic mobility on starch-gel and accounted for approximately 8, 70 and 22% respectively of the total neurophysin present in the gland. The assignment of the major rat posterior pituitary lobe protein as a neurophysin (neurophysin-II) is confirmed by its ability to bind oxytocin and [8-arginine]-vasopressin. A minimum molecular weight of 10056 was calculated for neurophysin-II from its amino acid analysis and based upon the presence of one molecule of methionine and one molecule of tyrosine per molecule of protein. The stoicheiometry of the neurophysins relative to the neurohypophysial hormones is discussed.

IN VITRO STUDIES OF THE RELEASE MECHANISM FOR VASOPRESSIN IN RATS

1966 ◽  
Vol 53 (4) ◽  
pp. 644-654 ◽  
Author(s):  
N. A. Thorn
Keyword(s):  
Release Mechanism ◽  
Posterior Pituitary ◽  
High Concentration ◽  
Vasopressin Release ◽  
Pituitary Glands ◽  
Posterior Pituitary Gland ◽  
Stimulation Period ◽  
Releasing Effect

ABSTRACT A study was made of the release of vasopressin activity from groups of isolated posterior pituitary hemilobes of rats, incubated in media with different ionic compositions and with different drugs added to the medium. Nicotine, amyl nitrite and ATP, which have been reported to cause a large in vivo release of hormone had no significant releasing effect in vitro. Caffeine too did not cause any release of hormone activity. About 5% of the vasopressin activity extractable from the isolated posterior pituitary hemilobes was released during stimulation with a high concentration of potassium in the medium. No more than this percentage could be mobilized during such a stimulation, even after further subdivision of the posterior pituitary glands, prolongation of the stimulation period or after increasing the calcium concentration in the medium five-fold. No release into the medium of vasopressin binding protein could be demonstrated during stimulation of vasopressin release. The results seem to be in agreement with the hypothesis that the release of vasopressin is intimately associated with arrival of impulses to the nerve endings in the posterior pituitary gland and that it takes place from a small pool of readily available hormone, presumably by dissociation of the hormone from the carrier protein.

scholarly journals ISOLATED NERVE ENDINGS (NEUROSECRETOSOMES) FROM THE POSTERIOR PITUITARY

1967 ◽  
Vol 34 (1) ◽  
pp. 185-205 ◽  
Author(s):  
Elliot Bindler ◽  
Frank S. Labella ◽  
Madhu Sanwal
Keyword(s):  
Density Gradient ◽  
Neurosecretory Cell ◽  
Density Gradient Centrifugation ◽  
Degradation Products ◽  
Gradient Centrifugation ◽  
Lactic Dehydrogenase ◽  
Complete Separation ◽  
Nerve Endings ◽  
Posterior Pituitary ◽  
Electron Microscopic

Subcellular fractions of the bovine posterior pituitary, including one composed almost exclusively of pinched-off nerve endings (neurosecretosomes), were characterized electron microscopically, hormonally, and enzymically. 15% of the nerve terminals in the gland were isolated as neurosecretosomes, as estimated from determinations of lactic dehydrogenase, a soluble, cytoplasmic enzyme. Neurosecretosomes were subdivided into three fractions by density-gradient centrifugation. The three subfractions, each shown to be nearly homogeneous populations of neurosecretosomes by means of electron microscopic and enzymic criteria, differed from each other in their vasopressin/oxytocin (VP/OT) ratios. The VP/OT ratio increased from the lightest to the densest fraction, indicating that VP is localized to denser and OT to lighter neurosecretosomes; similar results have been obtained previously for subfractions of neurosecretory granules (NSG). No morphological differences were apparent in neurosecretosomes among the three subfractions. Although complete separation of VP and OT was not achieved, the findings suggest that VP and OT are each stored in a different species of nerve ending and support the hypothesis that a given neurosecretory cell synthesizes, stores, and secretes only one of the peptide hormones. Microvesicles, 40–80 mµ diameter and contained in typical neurosecretory cell terminals, are believed to be degradation products of membrane ghosts of depleted NSG; electron micrographs indicative of this transformation are presented. A fraction rich in microvesicles, but containing some NSG membranes, was prepared by density-gradient centrifugation of an osmolysate of neurosecretosomes. Smaller, apparently nonneurosecretory nerve endings, lacking NSG but filled with small vesicles, are occasionally seen in sections from whole gland. The vesicles in these atypical posterior pituitary nerve endings may be true neurohumor-containing, "synaptic" vesicles.

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