Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport.

We have compared the retrograde axonal transport of horseradish peroxidase (HRP), to the retrograde transport of HRP conjugated with wheat germ agglutinin (WGA). Morphometric studies have shown that WGA-HRP conjugates were 40 times more sensitive than free HRP, in the tracing of retrograde connections from the rat submandibular gland to the superior cervical ganglion. Also, WGA-HRP was more sensitive than free HRP in the tracing of retrograde connections from the rat tongue to the hypoglossal nucleus. Our findings with WGA-HRP are consistent with the observations by Schwab et al. who reported (-125I) WGA is a highly sensitive retrograde tracer (Brain Research 152:145, 1978 (22)).     

Electron microscopic study of the orthograde axonal transport of horseradish peroxidase in the supraoptico-neurohypophysial tract of the rat

Summary Horseradish peroxidase (HRP) has been used as a protein tracer in order to visualize the ultrastructural sites of the orthograde transport of protein macromolecules in the hypothalamo-neurohypophysial tract of the rat. After a local injection of HRP within the supraoptic nucleus, the reaction product was observed: (1) mainly in tubules of the smooth endoplasmic reticulum in the more proximal part of the axons, and (2) in granules and microvesicles of the axon terminals. Observations on thick sections clearly showed the existence of a relationship between the smooth endoplasmic reticulum containing HRP and the labeled granules or microvesicles. These data are in good agreement with previous findings showing the existence of direct continuity between tubules of the smooth endoplasmic reticulum and a fraction of the neurosecretory granules and microvesicles. This evidence further reinforces the hypothesis that the latter organelles may possibly originate locally in the axons from the tubules of the smooth endoplasmic reticulum which may therefore be proposed as a possible vehicle for a non-granular intra-axonal transport of neurosecretory material in neurosecretory neurons.     

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB—H₂O₂ sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane—plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N—acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.     

A horseradish peroxidase study on the mammillothalamic tract in the rat

The mammillary projections to the anterior thalamic nuclei were investigated in the rat, using the horseradish peroxidase (HRP) method. Pars centralis of the medial mammillary nucleus projects to the medial portion of the ateromedial nucleus (AM). Pars medialis (Mm) of the medial mammillary nucleus sends fibers to the ipsilateral AM and sparsely to the medial portion of the contralateral side. The ventral and dorsal portions of Mm project to the anterior and posterior portions of AM, respectively. The pars latralis (Ml) and pars posterior (Mp) of the medial mammillary nucleus send fibers predominantly to the ipsilateral anteroventral nucleus and sparsely to the contralateral side. A slight difference between Ml and Mp projections was observed. The lateral mammillary nucleus projects bilaterally to the anterodorsal nucleus.     

Cholinesterase activity of capillaries in the rat brain. A light and electron microscopic study

Synopsis The presence of cholinesterase activity in association with capillaries of the central nervous system was investigated in the rat by means of both light and electron microscopic methods. Throughout most of the rat brain, the smaller blood vessels stain intensely for butyrylcholinesterase activity. In some areas, such as the commissural nucleus of the vagus and parts of the medial thalamus, the capillaries possess both acetylcholinesterase and butyrylcholinesterase activity. Blood vessels in those structures which lie outside the blood-brain barrier are completely devoid of cholinesterase activity. The electron microscope reveals that reaction product occurs within the matrix of the basement membrane, in the intermembranous space of the endothelial nuclear envelope and occasionally in the endothelial granular endoplasmic reticulum. It is suggested that the presence of cholinesterase within the basement membrane of brain capillaries is evidence of the role that the basement membrane may play in transfer mechanisms.     

A study of the two-way transport of horseradish peroxidase across the visceral pleura

Summary The authors report on a study of the transpleural transport of horseradish peroxidase after intrapleural and intracardiac application. Following intrapleural introduction, a retention of the marker on the apical membrane of mesothelial cells was observed, with subsequent transcellular transfer after incorporation into microvesicles. Following intracardiac injection, the marker moved out of the pulmonary capillaries across the endothelial vesicles and progressed to the pleural cavity across the intercellular spaces and mesothelial vesicles. With either route of injection, reaction product was noted in the basal lamina of the mesothelium, elastic membrane, alveolar macrophages and pneumocytes type II.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

A study of the two-way transport of horseradish peroxidase across the visceral pleura

The authors report on a study of the transpleural transport of horseradish peroxidase after intrapleural and intracardiac application. Following intrapleural introduction, a retention of the marker on the apical membrane of mesothelial cells was observed, with subsequent transcellular transfer after incorporation into microvesicles. Following intracardiac injection, the marker moved out of the pulmonary capillaries across the endothelial vesicles and progressed to the pleural cavity across the intercellular spaces and mesothelial vesicles. With either route of injection, reaction product was noted in the basal lamina of the mesothelium, elastic membrane, alveolar macrophages and pneumocytes type II.     

A light and electron microscopic study of osteodentin formation in the rat incisor after adriamycin administration

The effect of adriamycin (10 mg/kg body weight) on the rat incisor was investigated in 8-day-old animals at 9 days and 14 days after subcutaneous injection. The drug produced changes that were still present 14 days after administration. During this time osteodentin formation, which appeared to be the principal effect of the drug on the incisors, occurred to such an extent that in some regions of the teeth the pulp chamber was almost completely occluded. The formation of osteodentin began at the periphery of the pulp and gradually advanced towards the central region. Moreover, in some sections of the incisor the dentin layer was greatly reduced to a thin superficial layer, while osteodentin surrounded most of the pulp chamber. Cells that appeared to be differentiated pulp-mesenchymal cells were found within as well as on the surface of the irregular osteodentin matrix. Since this drug has been used in the treatment of childhood osteosarcomas, the possibility of dental abnormalities developing in these children cannot be overlooked.     

A light and electron microscopic study on the embryonic development of the rat carotid body.

The embryonic development of the rat carotid body was studied with electron microscopy. In the 11 mm embryo a cell aggregation consisting of undifferentiated cells and unmyelinated nerve fibers appears on the anterior wall of the third branchial artery. Granule-containing cells appear in the 12 mm embryo and continue to increase in number as the cellular aggregation increases in size and becomes separated from the wall of the third branchial artery. Synapse formation and the appearance of fenestrated capillaries occur almost simultaneously at the 17 mm stage. There are two types of synapses, one with membrane densification and vesicles clustered inside the nerve endings, the other with dense material and vesicles inside the granule-containing cells. At the 20 mm stage the undifferentiated cells send enveloping cytoplasmic processes toward adjacent granule-containing cells and the carotid body anlage displays rudimentary lobules.     

Differences between the endocytosis of horseradish peroxidase and its conjugate with wheat germ agglutinin by cultured fibroblasts

A covalent conjugate of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP) was used for a morphologic study of its adsorptive endocytosis by cultured human fibroblasts. Initial binding at 4°C of the conjugate was observed over the entire plasma membrane, including “coated” and smooth pits. Endocytosis of HRP and the WGA-HRP conjugate was observed in lysosomes, but only the conjugate was seen in a cisterna of the Golgi apparatus (GERL), and in adjacent coated vesicles.     

A light and electron microscopic study of sporulation in the myxomyceteStemonitis virginiensis

Summary The conversion of the plasmodium ofS. virginiensis into sporophores has been examined at both the light and electron microscopic levels. Particular attention has been paid to stalk and columella formation, capillitial formation, nuclear behavior during sporulation and spore formation. Both the stalk and columella are formed within the sporangial initial as intraprotoplasmic secretions. A portion of the capillitium arises directly from the columella while the remainder forms within an anastomosing system of tubular vacuoles. As spore cleavage begins the nuclei within the sporangium begin to divide mitotically. The protoplasmic content of the sporangium is first divided into small protospores which typically contain a single dividing nucleus. Following the completion of mitosis each of these segments cleaves into yet smaller segments which develop into spores. Meiosis occurs in the spores some 12–16 hours after cleavage.