Ultrastructural demonstration of neurohaemal contacts in the internal zone of the median eminence of the Mongolian gerbil (Meriones unguiculatus): correlation with synaptophysin immunohistochemistry

Electron microscopy of the median eminence (ME) of the Mongolian gerbil (Meriones unguiculatus) revealed that, unlike most other mammalian species, abundant neurohaemal contacts were present not only in the external zone (EZ), but also in the internal zone (IZ) up to the subependymal layer. In the IZ, nerve terminals with dense core vesicles and/or small clear vesicles abutted on the outer basal lamina of the perivascular space of portal capillaries, alternating with tanycyte processes. In addition to these neurohaemal contacts, several layers of vesicle-filled varicosities surrounded the portal vasculature. An analysis of serial thin sections showed that the latter varicosities could also reach the perivascular basal lamina or contact it through small extensions in other planes of section. Apparently at least some of the nerve terminals making neurohaemal contacts were en passant in nature. A correlative investigation of synaptophysin (a major integral membrane protein of small synaptic vesicles) immunoreactivity at the light microscopical level demonstrated a conspicuously dense immunostaining around portal capillaries in both EZ and IZ of the proximal and distal ME (neural stalk). Since this perivascular accumulation of immunoreactivity coincides precisely with the ultrastructural accumulation of vesicle-filled axons which establish numerous neurohaemal contacts, it is concluded that this pattern of synaptophysin immunostaining indicates sites of neurohaemal contacts at the light microscopical level. During postnatal development, the perivascular concentration of synaptophysin immunoreactivity in the IZ appeared concomitantly with the early postnatal expansion of long portal capillary loops into the IZ.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hypothalamo-hypophysial system of hypophysectomized rats

Summary The median eminence (ME) of hypophysectomized rats was studied by means of light and electron microscopy. Paraldehyde-fuchsin (PAF)-positive material is seen in the external zone (EZ) of the ME 2–5 days after the operation. Its amount gradually increases especially in the caudal part of the ME during the following few days. Some PAF-positive fibers make contact with the subependymally located blood capillaries. In the most caudal region of the recessus infundibuli they penetrate into the third ventricle. PAF-positive material decreases markedly from the ME of rats two months after hypophysectomy and exposure to a 1% salt load. Fibers of types A₁, A₂ and B containing granules of 120–220 nm, 100–150 nm and 80–100 nm in diameter, respectively, are seen in the EZ of the ME in hypophysectomized rats, although almost exclusively A₂- and B-type structures make contact with the primary portal capillaries in intact animals. All types of neurosecretory fibers establish contact with the subependymal nonfenestrated blood capillaries and penetrate the recessus infundibuli. Some neurosecretory terminals of different types make direct contact with the glandular cells of the pars tuberalis or are separated from them by a thin basal lamina. It is assumed that mainly neurosecretory fibers of types A₂ and B are permanently connected with the primary portal capillaries in the EZ of the ME in intact mammals, while the overwhelming majority of fibers of A₁-type shows ingrowth during the course of postoperative reparation. The possible physiological significance of the described changes is discussed.     

The hypothalamo-hypophysial system of the frog Rana temporaria

Summary The median eminence (ME) of the adult frog, Rana temporaria, was studied by means of electron microscopy including quantitative electron-microscopic autoradiography. In frogs captured in May and June numerous peptidergic neurosecretory fibres extending via the internal zone to the pars nervosa display large swellings containing few granules, mitochondria, neurotubules and cisternae of the smooth endoplasmic reticulum. In addition, few secretory globules up to 1.5 m in diameter occur in these varicosities. In animals collected during the autumn period many of these neurosecretory swellings filled with neurosecretory granules and polymorphic inclusions resemble Herring bodies. Three types of granule-containing neurosecretory fibres were observed in the external zone (EZ) of the ME of adult R. temporaria. Peptidergic A₁- and A₂-type fibres are characterized by granules 150–220 nm and 100–160 nm in diameter, respectively. Monoaminergic fibres of type B with granules approximately 100 nm in diameter represent 50% of all neurosecretory elements in the EZ of the frog ME; 12% of the total number of granule-bearing axons in the EZ actively taking up radiolabelled 5-hydroxytryptophan are thought to be serotoninergic terminals. Neurosecretory terminals of all types and glial vascular endfeet establish direct contacts with the perivascular space of the primary portal capillaries. Some neurosecretory terminals are separated from the lumen of the third ventricle by a thin cytoplasmic lamella of tanycytes. The possible physiological significance of this structural pattern is discussed.     

Ultrastructural study of neurotensin immunoreactivity in the superficial laminae of the dorsal horn of the rat

Neurotensin immunoreactivity was identified in cell bodies, dendrites, spines, axons, terminals and varicosities in superficial laminae of rat spinal cord with the electron microscope. Unlabeled terminals synapsed with neurotensin-immunoreactive cell bodies, dendrites and spines. Presynaptic terminals contained round or pleomorphic vesicles and generally made symmetrical contacts with medium-sized neurotensin-containing dendrites in outer lamina II, and asymmetrical or symmetrical contacts with large and small dendrites and spines in inner lamina II. Neurotensin immunoreactive axons were unmyelinated, and their terminals were presynaptic to unlabeled dendrites and spines in laminae I and II. Terminals contained small, round, clear vesciles (31 nm) and occasional large granular vesicles (78 nm). Contacts in outer lamina II were evenly distributed among dendrites of various sizes and spines, whereas the majority of labeled terminals in inner lamina II made contacts onto small dendrites and spines. These findings indicate that neurotensin effects in rat spinal cord are mediated by axodendritic synapses, and that neurotensin cells at the inner and outer borders of lamina II contact dendrites of efferent neurons or other interneurons in the dorsal horn.     

Immunocytochemical localization of TRH and autoradiographic determination of 3H-TRH-binding sites in the arcuate nucleus-median eminence of the rat

Immunocytochemical and autoradiographic localization of thyrotropin-releasing hormone (TRH)- and 3H-TRH-binding sites was studied in the arcuate nucleus-median eminence region of the rat. TRH-like immunoreactivity was found in dense granular vesicles (90-140 nm in diameter) in TRH-like immunoreactive nerve fibers and terminals. In the median eminence, the immunoreactive terminals were observed to be in direct contact with the perivascular basal lamina of the portal vessel and to form synaptoid contacts with tanycytes. In the arcuate nucleus, the immunoreactive terminals were often found to form axosomatic and axo-axonic, and/or axo-dendritic synapses. The uptake of tritiated TRH into the nerve fibers and terminals of the median eminence was also observed by autoradiography and the distribution and localization of silver grains in them were analyzed quantitatively by circle analysis. Thirty minutes after intraventricular infusion of 3H-TRH, radioactive labeling occurred in type-2 and 3-nerve fibers and terminals containing dense granular vesicles in the median eminence. It is therefore suggested that the neurons labeled after 3H-TRH infusion possess certain functions as physiological recognition sites or receptors for TRH.     

Allatostatin-like immunoreactivity in the abdomen of the locust Schistocerca gregaria

A polyclonal antibody against allatostatin 1 (AST-1) of cockroach Diploptera punctata was used to investigate the distribution of AST-like immunoreactivity within the abdomen of the locust, Schistocerca gregaria. In the abdominal ganglia, AST-like immunoreactivity was found in both cell bodies and neuropile. In ganglia 6 and 7, staining was found in serial homologous cell bodies in anterior dorsolateral and dorsomedial, and posterior ventrolateral and ventromedial locations. In the terminal ganglion, the numerous immunoreactive somata were smaller in size than those in the unfused ganglia. The combination of backfill experiments with immunocytochemistry showed that, in abdominal ganglion 7, one neuron of the ventromedian cell body cluster and two of the ventrolateral cluster innervated the oviduct, which itself was covered with a dense mesh of AST-immunoreactive varicosities. Combining electron microscopy with immunohistochemistry revealed AST-like immunoreactivity in dense-core vesicles located in neurohaemal-like terminals lacking structures normally found in synapses. A mesh of AST-immunoreactive varicosities was also found on the muscles of the spermatheca and the spermathecal duct. In addition, a mesh of strongly stained varicosities was present in the distal perisympathetic organs (neurohaemal organs in the abdomen) and on the lateral heart nerves (a putative neurohaemal release zone). These data indicate that AST is an important neuroactive substance that is probably involved in multiple tasks in the control of the locust abdomen.     

Distribution of synaptophysin immunoreactivity in guinea pig heart

The distribution of synaptophysin, an integral polypeptide of presynaptic vesicle membranes, was investigated in guinea pig heart by immunohistochemistry using monoclonal antibodies. Synaptophysin immunoreactivity was found in varicose nerve terminals in all regions of the heart. Dense networks of immunoreactive varicosities were found to surround the vasculature and to be located within the subendocardial layers of the atria and ventricles, the highest levels being seen in the innervation of the conductive system. As synaptophysin is probably a component of the presynaptic vesicles of all synapses, its use as a marker of all nerve terminals within the heart is proposed.     

Distribution of synaptophysin immunoreactivity in guinea pig heart

Summary The distribution of synaptophysin, an integral polypeptide of presynaptic vesicle membranes, was investigated in guinea pig heart by immunohistochemistry using monoclonal antibodies. Synaptophysin immunoreactivity was found in varicose nerve terminals in all regions of the heart. Dense networks of immunoreactive varicosities were found to surround the vasculature and to be located within the subendocardial layers of the atria and ventricles, the highest levels being seen in the innervation of the conductive system. As synaptophysin is probably a component of the presynaptic vesicles of all synapses, its use as a marker of all nerve terminals within the heart is proposed.Supported by the Deutsche Forschungsgemeinschaft (SFB 90) requests should be sent     

Intraventricular neuronal complex of the lamina terminalis of the mouse

An intraventricular neuronal complex has been identified with scanning and transmission electron microscopy at the base of the lamina terminalis of the mouse. The raspberry-shaped complex protrudes from a thickened bulge on the ependymal surface of the lamina terminalis or adjacent rostral floor of the third ventricle. Neurons and occasional ependymal cells cover the surface of the complex. Its core is made up of neurons, ependymal cells, and neuronal processes, which are usually compactly arranged. The core is continuous, through a breach in the ependymal layers, with the subependymal neuropil of the lamina terminalis. Within the core of the complex are large numbers of axodendritic synapses. Axonal varicosities and synaptic terminals are filled with vesicles and mitochondria. Synaptic endings have one of two populations of vesicles: exclusively clear, small, round or flattened vesicles. In view of the known structural and functional characteristics of the lamina terminalis, it is possible that the neuronal complex may participate in neurohormonal regulatory systems of the hypothalamus and hypophysis or in the network of circumventricular organs mediating angiotensin effects.     

Glutamate immunoreactivity is enriched over pinealocytes of the gerbil pineal gland

Summary Mammalian pinealocytes have been shown to contain synaptic-like microvesicles with putative secretory functions. As a first step to elucidate the possibility that pinealocyte microvesicles store messenger molecules, such as neuroactive amino acids, we have studied the distributional pattern of glutamate immunoreactivity in the pineal gland of the Mongolian gerbil (Meriones unguiculatus) at both light- and electron-microscopic levels. In semithin sections of plastic-embedded pineals, strong glutamate immunoreactivity could be detected in pinealocytes throughout the pineal gland. The density of glutamate immunolabeling in pinealocytes varied among individual cells and was mostly paralled by the density of immunostaining for synaptophysin, a major integral membrane protein of synaptic and synaptic-like vesicles. Postembedding immunogold staining of ultrathin pineal sections revealed that gold particles were enriched over pinealocytes. In particular, a high degree of immunoreactivity was associated with accumulations of microvesicles that filled dilated process terminals of pinealocytes. A positive correlation between the number of gold particles and the packing density of microvesicles was found in three out of four process terminals analyzed. However, the level of glutamate immunoreactivity in pinealocyte process endings was lower than in presumed glutamatergic nerve terminals of the cerebellum and posterior pituitary. The present results provide some evidence for a microvesicular compartmentation of glutamate in pinealocytes. Our findings thus lend support to the hypothesis that glutamate serves as an intrapineal signal molecule of physiological relevance to the neuroendocrine functions of the gland.     

Ontogenetic appearance of somatostatin-containing nerve terminals in the median eminence of rats

Summary The development of immunoreactive (ir) somatostatin-containing nerve terminals in the rat median eminence (ME) has been examined electron-microscopically. Nerve fibers containing ir particles scattered throughout the axoplasm are first seen in the external layer of the ME on day 18.5 of gestation, and, on day 21.5 appear to terminate on the basement membrane of the perivascular space of the portal vessels. After birth, the fiber terminals contain several membrane-limited granules, which are labeled with ir PAP particles. Ultrathin, Epon-embedded sections of ME, treated by the protein A gold-labeling method for somatostatin, demonstrate positively labeled granules in the nerve fibers in the postnatal ME, but in the prenatal tissue, no specific gold-labeling is found. These findings show that, in the external layer of the ME, somatostatin storing occurs in the granules in the axonal terminals after birth.     

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.     

Secretory cells in the nucleus pulposus of the adult human intervertebral disc. A preliminary report

A light microscopical study was conducted to ascertain the type of cells in the nucleus pulposus of the adult human intervertrebral disc. Three lumbar intervertebral discs were removed from each of 15 male and female adults at autopsy (ages ranged from 19 to 62 years). The tissue was fixed in formalin, decalcified in formic acid, dehydrated in a graded series of ethanol, embedded in paraffin, and serially sectioned at 7-10 micron. Tissue sections were affixed to albuminized glass slides and stained either by hematoxylin and eosin or hematoxylin and Van Gieson's stain. The cells of the bulk of the nucleus pulposus consisted of chondrocytes and a few fibroblasts; however, the subchondral matrix of the nucleus pulposos contained numerous stellate cells with (from 1 to 8) unusually long (up to 80 micron) primary cytoplasmic processes that often branch into secondary processes. The cell processes contained cytoplasmic varicosities at various intervals along their lengths; and their endings often expanded into bulbous, vesicle-filled process terminals. The surrounding extracellular matrix usually contained numerous, vesicle-filled, eosinophil matrix bodies. Morphological similarities of cytoplasmic varicosities, process terminals, and matrix bodies, as well as the apparent budding of process terminals, suggest that these previously unidentified cells are secreting an unknown matrix component into the subchondral matrix of the nucleus pulposus of the adult human.     

The interrelations of the lymphatic capillaries with the nerve structures in the wall of the small intestine]

The character of interrelations of nervous structures and the lymphatic capillary walls has been studied in cats. Under the light microscope twisted nervous fiber terminals of the intestinal neuron dendrites have been revealed around the lymphatic capillaries. Electron microscopical investigation has not revealed any specialized contacts of the nervous terminals and the lymphatic capillary walls. The receptors and terminals of axons do not situate nearer than 10 nm from the latter. According to the structure of synaptic vesicles among the axonal terminals next to the lymphatic capillary walls cholinergic, adrenergic and purinergic ones are described. The influence of the nervous system to the function of the small intestine lymphatic capillaries is mediated via the precapillary space. The neuromediators from the axonal terminals get into it owing to absence of neurolemmocytic membranes around them.     

Connective tissue in the perivascular spaces of subependymal capillaries of the spinal cord in the rabbit]

The normal structure of the subependymal capillaries and venules of the spinal cord was studied in rabbit. The endothelial cells of the capillaries and venules are surrounded by an irregularly formed perivascular space, about 0.5 to 3.3 micrometer wide, which is delimited by an endothelial and glial basal lamina. The space contains a framework of collagen fibers. A period-acid-bisulfit-aldehydthionine-method (Specht) permits to find the perivascular connective tissue lightmicroscopically, while they can be identified by electron microscopy. The significance of the perivascular connective tissue is open to discussion. Structural and functional problems have been reviewed in this context.     

Substance P-containing axon terminals in the mucosa of the human urinary bladder: pre-embedding immunohistochemistry using cryostat sections for electron microscopy

The ultrastructure of substance P (SP)-containing axon terminals in the mucosa of the human urinary bladder was studied. Numerous SP-immunoreactive varicose nerve fibers were seen in the lamina propria, and most of them ran freely in the connective tissue. Many SP-immunoreactive nerve fibers were observed beneath the epithelium, and perivascular SP-immunoreactive nerves were also found in the submucosal layer. We observed a total of 305 SP-immunoreactive (IR) axon terminals, of which most (89.6%) were free nerve endings at the ultrastructural level; the rest of the SR-IR axon terminale were seen in the vicinity of the epithelium and blood vessels in the lamina propria. Varicose regions of SP-IR axon terminals contained large granular and small agranular synaptic vesicles, and most of them partially lacked a Schwann cell sheath. In some SP-IR varicosities, synaptic vesicles were concentrated in the region without any Schwann cell sheath. Long storage (for more than 1 month) of fixed-tissue pieces in sucrose before freezing has improved the ultrastructure of cryostat sections in pre-embedding immunohistochemistry. Trypsin digestion for the purpose of exposing antigenic sites was also employed before applying the first antiserum.     

Immunocytochemical study of the GABAergic innervation of the mouse pituitary by use of antibodies against gamma-aminobutyric acid (GABA)

Summary The GABAergic innervation of the mouse pituitary, including the median eminence, was studied at light microscopic and ultrastructural levels by use of a pre-embedding immunocytochemical technique with antibodies directed against GABA. In the median eminence, a high density of GABA-immunoreactive fibers was found in the external layer where the GABAergic varicosities were frequently observed surrounding the blood vessels of the primary capillary plexus. In the internal and subependymal layers, only few fibers were immunoreactive. The intense labeling of the external layer was observed in the entire rostro-caudal extent of the median eminence. In the pituitary proper, a dense network of GABA-immunoreactive fibers was revealed throughout the neural and intermediate lobes, entering via the hypophyseal stalk. The anterior and tuberal lobes were devoid of any immunoreactivity. The GABA-immunoreactive terminals were characterized in the median eminence, and in the intermediate and posterior lobes at the electron-microscopic level. They contained small clear vesicles, occasionally associated with dense-core vesicles or neurosecretory granules. In the intermediate lobe they were seen to be in contact with the glandular cells. In the posterior lobe and in the median eminence, GABA-immunoreactive terminals were frequently located in the vicinity of blood vessels. These results further support the concept of a role of GABA in the regulation of hypophyseal functions, via the portal blood for the anterior lobe, directly on the cells in the intermediate lobe, and via axo-axonic mechanisms in the median eminence and posterior lobe.     

Synaptic and nonsynaptic release of neuromediators in the central nervous system

Different types of release site were studied ultrastructurally with tannic acid and immunohistochemical techniques in the central nervous system (CNS) of the invertebrate pond snail Lymnaea stagnalis and in two neuromediator rich core regions in the CNS of the rat, viz., the median eminence (ME) and the mesencephalic central grey substance (MCG). In the CNS of the snail, release of the contents of the secretory granules could be clearly demonstrated in (1) neurohaemal axonterminals, (2) synapses and (3) in nonsynaptic release sites: neuronal processes without morphological synaptic specializations. In the ME, release of secretory products by exocytosis was found in neurohaemal axonterminals in the external part of the palisade layer and in nonsynaptic release sites in all other layers of the median eminence. It was found that oxytocine and vasopressin were released by exocytosis into the extracellular space from such (preterminal) nonsynaptic release sites. Serial section analysis revealed three types of fibre in the MCG, viz. (1) varicose fibres that made synaptic contacts with MCG dendrites on every varicosity, (2) fibres with two types of varicosity, viz. synapse-bearing varicosities and varicosities without synaptic specializations, and (3) varicose fibres without any synaptic specializations. It has been discussed that the nonsynaptic release sites in the CNS of the snail Lymnaea stagnalis, and the nonsynaptic varicosities in the rat brain are the morphological correlates of nonsynaptic communication in the CNS. The results further indicate that particular peptidergic neuromediators are released from such nonsynaptic varicosities, and may reach via the extracellular space receptors located at some distance.     

Synaptophysin immunoreactivity in the mammalian endocrine pancreas

Summary Synaptophysin, a major membrane glycoprotein of small presynaptic vesicles in neurons, has also been found in microvesicles of endocrine cells, e.g., of the endocrine pancreas. In the present study, the endocrine pancreas in 9 mammalian species (man, dog, mink, bovine, rabbit, guinea pig, rat, mouse, gerbil) has been investigated immunohistochemically for synaptophysin immunoreactivity. Synaptophysin-positive cells have been identified and localized on semithin plastic sections. Our study demonstrates that, in all species examined, all pancreatic endocrine cell types are consistently synaptophysin-positive independent of their location within the tissue, or the conditions of tissue processing. In addition, a few cells that cannot be hormonally identified show synaptophysin immunoreactivity. Hence, synaptophysin appears to be a regular constituent of all pancreatic endocrine cells in mammals. In several species, a subpopulation of endocrine cells, consisting of glucagon-containing and/or pancreatic-polypeptide-containing cells, exhibits a significantly higher degree of synaptophysin immunoreactivity. In the gerbil, this heterogeneity can readily be detected from the day of birth onwards. Our findings indicate that closely related endocrine cell types may differ with respect to the content of synaptophysin.     

Immunomicroscopical observations on the nervous system of adult Eudiplozoon nipponicum (Monogenea: Diplozoidae)

Neuronal pathways have been examined in adult Eudiplozoon nipponicum (Monogenea: Diplozoidae), using cytochemistry interfaced with confocal scanning laser microscopy, in an attempt to ascertain the status of the nervous system. Peptidergic and serotoninergic innervation was demonstrated by indirect immunocytochemistry and cholinergic components by enzyme cytochemical methodology; post-embedding electron microscopical immunogold labelling revealed neuropeptide immunoreactivity at the subcellular level. All three classes of neuronal mediators were identified throughout both central and peripheral elements of a well-differentiated orthogonal nervous system. There was considerable overlap in the staining patterns for cholinergic and peptidergic components, while dual immunostaining revealed serotonin immunoreactivity to be largely confined to a separate set of neurons. The subcellular distribution of immunoreactivity to the flatworm neuropeptide, GYIRFamide, confirmed neuropeptide localisation in dense-cored vesicles in the majority of the axons and terminal varicosities of both central and peripheral nervous systems. Results reveal an extensive and chemically diverse nervous system and suggest that pairing of individuals involves fusion of central nerve elements; it is likely also that there is continuity between the peripheral nervous systems of the two partner worms.