Nongranular vasopressin synthesis and transport in early stages of rehydration

Summary The vasopressin system of the rat was examined in the course of the first 12 h of rehydration after prolonged thirst at light and electron microscopic levels and by use of the peroxidase anti-peroxidase (PAP) method. Light microscopically, the median eminence was the only part of the system that not only displayed distinct differences between animals of different rehydration times but also showed a characteristic pattern of immunohistochemical reactivity in its rostro-caudal distribution. Ultrastructurally, in the perikarya a maximal labeling of the rough endoplasmic reticulum was observed after 2 h of rehydration, whereas an extensive labeling of the enlarged Golgi zones was attained after 4 h of resupplying water. A labeling of the intercellular clefts in the basal glial labyrinth of the supraoptic nucleus (and to a lesser degree in the subependymal neuropil adjacent to the paraventricular nucleus) was increased 30 min after the onset of drinking, as compared with water-deprived animals; it decreased slightly after 12 h of rehydration. The filling of the swollen fibers by increasing amounts of labeled axoplasmic reticulum, evident in the nuclear areas already after 30 min of water supply, begins in the median eminence after 2 h of rehydration and is fully developed after 4 and 8 h. Corresponding results hold true for the neural lobe but are somewhat delayed in comparison to the findings in the median eminence. The discussion considers (i) synthesis and transport of nongranular vasopressin within the axoplasmic reticulum, and (ii) release not only from the neural lobe but also from the nuclear areas and from the fibers of the median eminence.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung Volkswagenwerk     

Neurohormones in the intercellular clefts and in glia-like cells of the rat brain

Summary With the aid of electron microscopic immunocytochemistry following the application of antisera against somatostatin and luliberin (LRF), a labeling of the intercellular clefts in different areas of the brain was observed. This labeling is especially conspicuous near the basal pole of the cuboidal ependymal cells, but is also generally present in all regions containing neurohormone-producing perikarya or their processes (for example, the preoptic area, the basal ganglia and the cortex).Furthermore, in all these regions displaying labeled intercellular clefts, glialike cells and sparsely ciliated ependymal cells are found, the secondary lysosomes of which exhibit an immunoreactivity resembling that observed in the intercellular clefts.As sources of the immunoreactive material the following possibilities are discussed: (i) perikarya producing somatostatin or LRF, situated in the wall of the third ventricle and sending fibers between the cuboidal ependymal cells, (ii) hypothalamic and extrahypothalamic projections of both peptidergic systems, and (iii) in the case of somatostatin, immunoreactive perikarya in the cortex.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung VolkswagenwerkDedicated to Professor Walter Kirsche on the occasion of his 60th birthday     

Evidence for a transcellular cisternal route across the caecal epithelium of an insect

Summary The cells of the mesenteric caeca in the midgut of certain insects possess a labyrinth of transepithelial cisternae. Their existence can be seen in thin sections of lanthanum-incubated tissue, where the tracer enters not only the intercellular clefts but also membranous cisternae which are inpocketings from, and, in continuity with, both the lateral clefts and basal membrane. These infoldings, which are numerous, run from the basal or lateral surfaces into the perinuclear region of the cells, where they are found, laden with lanthanum, as smooth cisternae or vesicles in the peripheral cytoplasm near the plasma membrane. These can be followed in serial sections and are quite distinct from other sub-surface cisternae of the lateral borders which are studded with ribosomes on the cytoplasmic surface. Near the luminal surface, tracer-laden structures in the form of vesicles and granules become increasingly predominant over those in the form of cisternae. Freeze-fracture replicas confirm the above observations, in that the plasma membrane of the intercellular cleft can be characterized as such unequivocally, since it exhibits smooth septate junctional E face grooves and P face ridges. Lateral infoldings, cisternae and vesicles can be seen arising directly from these junction-bearing membranes. The transepithelial cisternae and vesicles may be the morphological basis of an insect transcellular transport system, comparable to the tubulocisternal endoplasmic reticulum present in the transporting secretory and absorptive epithelia of vertebrate tissues. However, in insect midgut caecal epithelia, the cisternae appear to be, albeit presumably transiently, in direct continuity with the extracellular space, forming a plasma membrane reticular system which seems not to be the case with the tubulo-cisternal endoplasmic reticulum which terminates in subsurface cisternae.     

The brain's interstitial clefts and their glial walls

The heterogeneous contents of the CNS interstitial clefts and the configuration of their astrocytic walls may be regionally variable. Astrocytic processes of the glia limitans, in normal midbrain and in astroglial scars, form thin, parallel, concentric sheets comprising the walls of narrow interstitial clefts. There is a critical thickness of about 20 to 30 nm, below which astrocytic cell process or those of the fibroblast-like cells in the meninges, do not invaginate to form transcytotic vesicles. Large hydrophilic solutes cannot, therefore, pass across the thin portion of a cell process. Consequently, (a) the diffusion and convection paths of interstitial fluid and solutes are lengthened, (b) a solute will remain within the interstitial cleft between thin lamellae for a relatively long time and (c) if a ligand does bind to its receptor on the thin process's cell membrane, there can be no receptor-mediated transcytosis at that site. Interstitial clefts, themselves, vary in size, shape and content, including extracellular matrix and basal lamina. A common constituent of basal lamina and extracellular matrix, presumably including that at ependymal, astroglial and endothelial interfaces of the CNS, is heparan sulfate proteoglycans. As in other organs, these proteoglycans may store growth factors, growth inhibitors, cytokines and other modulators which can then be released enzymatically during, e.g., regeneration. Exogenous heparan sulfate proteoglycan might serve as a natural, intermittent-release matrix for delivery of trophic factors.     

Transport of peroxidase through the midgut epithelium of Glossina M. Morsitans (diptera, glossinidae)

The peritrophic membrane (pm) of teneral female tsetse flies, Glossina morsitans morsitans, did not extend to the full length of the midgut 1-12 hr after emergence. The ingested blood did not reach the posterior part of the midgut (p-part), and the crop still contained food 12 hr after feeding. In these flies, the p-part contained the remains of the larval gut, the meconium, and bacteria. Ferritin molecules fed to tsetse females together with human serum were only found in the endoperitrophic space of the gut. This electron-dense tracer did not penetrate and cross the pm. On the other hand, ingested peroxidase passed the pm, and was transported through intercellular clefts, the basal labyrinth and the basal lamina to the hemolymph. This uptake was observed in the anterior part and to a smaller extent in the middle part of the midgut within 2 hr after feeding. Peroxidase was incorporated from the hemolymph into fat body cells, where it was found 2 hr and later after feeding. Pinocytosis of the tracer molecules, as an additional intracellular pathway to the intercellular route of transport, could not be demonstrated.     

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.     

The functional and structural border of the neurohemal region of the median eminence

Summary In stressed rats the tanycytes of the ventrolateral wall of the third ventricle exhibit by light microscopic immunohistochemistry a positive staining for neurohormones which is distinctly limited to the distal perivascular end of the tanycyte process. Since by electron microscopic immuncytochemistry the tanycyte cytoplasm does not show any reaction product, the light microscopic reaction most likely results from a labeling of the intercellular space in the direct vicinity of the subendothelial cleft. Whether this subendothelial space is permeable to neurohormones was tested by injection of HRP¹. In the region of the arcuate nucleus 30 min after intravenous application, the marker is affixed to the membranes of the perivascular tanycyte processes in the subendothelial cleft of capillaries possessing non-fenestrated endothelia. Occasionally, HRP penetrates for a short distance between the tanycytes. Then the labeling of the intercellular cleft ends abruptly. Here, several parallel ridges of tight junctions between the perivascular distal tanycyte processes are found by the freezeetching technique. Since HRP cannot reach the subendothelial clefts of this region by passing through capillary walls due to the presence of a blood-brain barrier, it is suggested that the marker penetrates from the median eminence this far via the subendothelial extracellular space. It is prevented from spreading further by the tight junctions of the perivascular tanycyte endings. The same way may be taken by the neurohormones. Hence, a border area exists adjacent to the dorsolateral aspect of the neurohemal region of the median eminence where the tanycytes isolate the neuropil from the cerebrospinal fluid not only by their apical tight junctions, but also by basal tight junctions from the subendothelial cleft. This communicates with the perivascular space of the portal vessels.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung VolkswagenwerkDedicated to Professor Dr. R. Ortmann on the occasion of his 65th birthday.The skilful technical assistance of Miss K. Bielenberg, Mrs. A. Hinz and Mrs. H. Prien is thankfully acknowledged     

Ultrastructure of intestinal and gall-bladder epithelium in the teleost Gasterosteus aculeatus L., as related to their osmoregulatory function

Summary Intestinal and gall-bladder epithelial cells in sticklebacks have been examined in ultrathin sections and freeze-etch replicas. Enterocytes throughout the intestine appear to have a well-developed basal labyrinth similar to that of renal tubular cells, consisting of baso-lateral infoldings closely associated with numerous mitochondria. The lumen inside these intracellular membranes is continuous with the intercellular space via pores. Such a membrane system is also present in the epithelial cells lining the gall bladder, distinguishing them from gall-bladder cells of higher vertebrates. Morphometric analysis indicates that the basal labyrinth of enterocytes in the posterior part of the intestine increases markedly in both sexually mature males and androgen-treated females. This does not occur in the anterior part or gall bladder. In sticklebacks, androgens cause reduced urine excretion and enhanced fluid release via the anus. We conclude that the cells lining the intestine and gall bladder possess an extensive basal labyrinth that may function as a backward channel system, enabling fluid to be produced in the intestine of fish. The androgen-induced increase in the extent of the basal labyrinth in the posterior part of the intestine may be related to the enhanced rate of intestinal fluid excretion observed in sexually mature male sticklebacks.     

The epithelium of the middle ear in the guinea pig

Summary An electron microscopic study of aldehyde and osmium fixed normal guinea pig middle ear epithelium was made. Numerous branching microvilli occur between the cilia of the ciliated cells. The granules of the secretory cells are always surrounded by a membrane, and they vary in their content of electron dense substance. Half desmosomes are frequent in basal cells. The squamous epithelial cells of the bulla contain few microvilli and pinocytoric invaginations. In the basal part of the squamous epithelium dilations of the intercellular clefts often occur. The luminal part of the intercellular clefts are closed by multiple tight junctions.     

Fine structure of supporting cells in the lateral-line canal-organ of Fundulus

Phase and electron microscopic studies have revealed that the supporting cells of the lateral-line canal-organ of Fundulus heteroclitus have a fine structure characterized by (1) mitochondria and vacuoles in the apical zone, (2) granular endoplasmic reticulum surrounding a core of Golgi complex in the middle zone, and (3) an indented nucleus in the basal zone. In the middle and basal zones, wide intercellular spaces separate the adjacent supporting cells, whose plasma membranes form long flap-like interdigitations across the intercellular spaces. Secretion of subcupular fluid and cupula, metabolic regulation through the intercellular spaces, and rigid support for the organ are discussed as possible metabolic and mechanical functions of the supporting cells essential for the lateral-line canal-organ.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

Ciliated neurons and different types of synapses in anterior hypothalamic nuclei of reptiles

Summary The magnocellular paraventricular and supraoptic nuclei and the parvocellular preoptic and periventricular nuclei have been studied by light and electron microscopy in Emys orbicularis, Lacerta agilis and Elaphe longissima. The ultrastructure of cerebrospinal fluid (CSF)-contacting neurons was described in the preoptic and periventricular nuclei of Emys and Lacerta species. Single 9×2+0 cilia similar to those of the CSF-contacting dendritic terminals were found on perikarya of non CSF-contacting nerve cells, in all four investigated nuclei. The cilia project from funnel-like invaginations of the perikarya into the intercellular space. In the neurons of the nuclei studied, granular vesicles were found, their size being mainly 1,600 Å in the paraventricular nucleus, about 1,800 Å in the supraoptic nucleus, 1,100 Å in the periventricular nucleus and 800 Å, or up to 1,250 Å in the preoptic nucleus. In general, the neurons possess synapses of the axo-somatic, axo-somatic spine, axo-dendritic and axo-dendritic spine types. In the supraoptic nucleus, multiple interdigitated synapses were observed. Presynaptically, either synaptic vesicles only, or synaptic vesicles and dense core vesicles of different sizes (600 to 800 Å, about 1,100 Å, 1250 Å, and up to 2,000 Å) were found. It is discussed whether the above described 9×2+0 cilia may represent some kind of hypothalamic sensory structure that earlier physiological studies postulated to exist. The ciliated hypothalamic perikarya are considered by the authors to be a more differentiated form of the CSF-contacting neurons. The different types of synapses indicate multilateral connections of the nerve cells of the nuclei studied.Dedicated to Prof. Dr. Berta Scharrer on the occasion of her 70th birthday     

Light- and electron microscopic localization of vasopressin or a vasopressin-like substance in the neurons of the rat suprachiasmatic nucleus

Summary In the suprachiasmatic nucleus of the rat light microscopic immunostaining for vasopressin reveals a distribution pattern of the immunoreactive material different from that known for the supraoptic nucleus. Among non-stained neurons positive-reacting perikarya display a cap- or tiplike labeling. The area of the suprachiasmatic nucleus is marked by delicate vasopressin-positive fibers. At the ultrastructural level the reaction product, after incubation with anti-vasopressin, is localized in small elementary granules unevenly distributed over the cytoplasm. Groups of axons containing specifically labeled granules contact non-reacting fibers.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung Volkswagenwerk     

ULTRASTRUCTURAL STUDIES ON THE PERMEABILITY OF THE MESOTHELIUM TO HORSERADISH PEROXIDASE

Peritoneal mesothelium was exposed for 2–60 min to solutions of horseradish peroxidase by incubation in vitro, or after intraperitoneal injection in vivo. Peroxidase was localized, with the electron microscope in the intercellular clefts of the mesothelium, often along their entire lengths, in vesicles adjoining or contiguous with the clefts, and along the peritoneal and basal surfaces of the cell, and also in intracytoplasmic vacuoles. The intercellular junctions of peroxidase-treated mesothelium did not differ from those of controls: open and closed junctions were present in both groups. Intercellular localization was also obtained when the mesothelium was exposed to peroxidase during or after fixation. Although intracellular absorption of peroxidase and its incorporation into larger vacuoles were observed, there was no clearcut evidence of vesicular transport across the mesothelium in these experiments. These findings are consistent with physiologic data which postulate that mesothelial transport can be accounted for, at least in part, by passive diffusion through a system of pores, and they suggest that these pores are located in the intercellular clefts.     

Access and distribution of exogenous substances in the intercellular clefts of the rat adenohypophysis

In model experiments with the use of horseradish peroxidase (HRP), two pathways of transport of substances to the adenohypophysis were studied, as well as the distribution of the tracer in the latter organ. The first pathway allows the tracer to penetrate from the intercellular milieu of the median eminence below the meningeal sheath covering the adenohypophysis to the surface of the pituitary gland. The second pathway transports the tracer via the capillaries of the hypophysial portal circulation to the interior of the glandular parenchyma. These results show (i) that the meningeal sheath establishes a barrier between the hemal milieu of the pituitary and the hemal milieu of the general circulation, and (ii) that the tracer reaching the adenohypophysis via both routes is found in the intercellular clefts of the glandular parenchyma only to a limited extent. By means of conventional electron microscopy, intercellular contacts between hormone-producing adenohypophysial cells are observed resembling focal tight junctions. Between the membranes of entwined processes of stellate cells, only small maculae adhaerentes are found. Freeze-etch studies on unfixed adenohypophyses reveal zonulae occludentes between the durafacing layers of the meningeal sheath and focal maculae occludentes between parenchymal cells. Additional tissue-culture experiments with adenohypophysial cells directly exposed to HRP reveal a gradual cessation of the labeling process in the intercellular clefts in accord with the observations from the in-vivo experiments, as well as intercellular focal tight junctions between individual hormone-producing cells.     

Histochemical studies on the morphology of the Golgi apparatus in the neurons of supraoptic and paraventricular nuclei of the normal and dehydrated rabbit (application of the thiamine pyrophosphatase method)

Summary Detailed histochemical studies have been conducted on the morphology of the Golgi apparatus by applying the thiamine pyrophosphatase technique (Novikoff and Goldfisher, 1961) to the neurons of supraoptic and paraventricular nuclei of normal and dehydrated rabbits. The neurons in both nuclei were classified into five categories on the basis of the morphology of the Golgi apparatus. The number of cells in individual categories were counted to evaluate the percentage of each category in the whole nucleus.Neurons have many vesicles which show the tendency to form clusters. Such clusters are present also in the basal bodies. The Golgi apparatus is localized near one side of the nucleus in many neurons. The neurons indicate phasic activity of resting, anabolic and catabolic stages under normal conditions.During dehydration, the Golgi apparatus went through the three stages of network formation, the increase of the budding-off process and later on disintegration. The supraoptic nucleus reacted to the TPPase test more severely than the paraventricular nucleus, whereas the former went through the stages more slowly than the latter. The paraventricular nucleus also revealed sensitivity to osmotic stress.     

Submicroscopic analysis of the microcirculatory system of the pancreas in several vertebrates]

The metabolic link of the microcirculatory system of the exocrinous part of the pancreas studied electron microscopically in the frog, chicken and rat has a general plan of the structure. It consists of capillaries, pericapillary gap and intercellular clefts of glandular cells connected with it. But in the frog and chicken the adventitional layer was found to be absent from the blood capillary wall, the luminal surface of endothelial cells was increased. The width of the basal layer and intercellular clefts in the rat was less than in other objects. The existence of cytoplasmic spiculae of exocrinous pancreocytes in the pancreas of different vertebrates allows to consider them as an element of the exocrinous part microcirculatory system.     

Noradrenaline as a putative neurotransmitter mediating hypotension—induced FOs—like immunoreactivity in the supraoptic nucleus of the rat

Hemorrhage or hypotension induces extensive Fos-like immunoreactivity in the magnocellular neurosecretory cells in the supraoptic nucleus of the hypothalamus in rat,especially in the vasopressin neurons.The present study was to explore the neurotransmitter mediating this effect,Microinfusion of the alpha-adrenergic blocker into the supraoptic nucleus reduced the hypotension-induced FOs.whereas beta-antagonist did not affect it significantly.Alaha1-and alpha2-antagonist,prazosin and yohimbine,both reduced the Fos-Positive cell counts.However,the effective dosage of yohimbine was much larger,Alpha1-agonist,methoxamine,induced abundant Fos-like immunoreactivity in the vasopressin cells in this nucleus,while beta-and alpha2-agonist did not elicit such effect.Administration of the noradrenergic re-uptake inhibitor desipramine,to this nucleus to locally accumulate the spontaneously released noradrenaline from the nerve terminals also induced Fos expression,mostly in the vasopressin cells.     

Bystander attenuation of neuronal and astrocyte intercellular communication by murine cytomegalovirus infection of glia

Astrocytes are the first cells infected by murine cytomegalovirus (MCMV) in primary cultures of brain. These cells play key roles in intercellular signaling and neuronal development, and they modulate synaptic activity within the nervous system. Using ratiometric fura-2 digital calcium imaging of >8,000 neurons and glia, we found that MCMV-infected astrocytes showed an increase in intracellular basal calcium levels and an enhanced response to neuroactive substances, including glutamate and ATP, and to high potassium levels. Cultured neurons with no sign of MCMV infection showed attenuated synaptic signaling after infection of the underlying astrocyte substrate, and intercellular communication between astrocytes with no sign of infection was reduced by the presence of infected glia. These bystander effects would tend to cause further deterioration of cellular communication in the brain in addition to the problems caused by the loss of directly infected cells.     

Effect on vasopressin release of microinjection of cholinergic agonists into the rat supraoptic nucleus.

It is likely that central cholinergic pathways to the paraventricular and supraoptic nuclei participate in the control of vasopressin release. We have shown previously that this is due, in part, to activation of muscarinic, but not nicotinic, receptors in the paraventricular nucleus. There is, however, reason to believe that this cholinergic effect in the supraoptic nucleus may be the result of activation of nicotinic receptors. To test this possibility, we have studied in conscious unrestrained rats the effect of microinjection of muscarinic and nicotinic agonists into the supraoptic nucleus on vasopressin release, mean arterial blood pressure, and heart rate. Under ether anesthesia, a stainless steel guide cannula was placed in the supraoptic nucleus 5-7 days before the experiment, and femoral, arterial, and venous catheters were implanted 1 day before the experiment. Microinjection of nicotine into the supraoptic nucleus at doses of 1 and 10 micrograms resulted in transient increases in the plasma vasopressin concentration that were 7-fold and 11-fold greater, respectively, than control values at 3 min. There were also small transient increases in mean arterial blood pressure, but heart rate was unchanged. The microinjection of 2 and 20 ng of oxotremorine, a muscarinic agonist, into the supraoptic nucleus had no effect on the plasma vasopressin concentration, mean arterial blood pressure, or heart rate. These doses of oxotremorine were previously shown to have potent stimulatory effects on vasopressin release when microinjected into the paraventricular nucleus. These findings suggest that the central cholinergic stimulation of vasopressin release is due, in part, to activation of muscarinic receptors in the paraventricular nucleus and nicotinic receptors in the supraoptic nucleus.