Distribution of neurotensin-like immunoreactive cell bodies and fibers in the brainstem of the adult male cat

We studied the distribution of cell bodies and fibers containing neurotensin in the brainstem of the cat using an indirect immunoperoxidase technique. A high or moderate density of immunoreactive perikarya was found in the interpeduncular nucleus, inferior colliculus, nucleus of the brachium of the inferior colliculus and in the lateral tegmental field. Moreover, a high density of neurotensin-immunoreactive fibers was observed in the periaqueductal gray, locus coeruleus and in the marginal nucleus of the brachium conjunctivum. The interpeduncular nucleus, nucleus of the solitary tract and the dorsal motor nucleus of the vagus contained a moderate density of immunoreactive fibers.     

Fine structure of ependymal cysts in and around the area postrema of the rat

Summary Peculiar cells forming cysts were observed in the area postrema and sometimes also in the choroid plexus and the tela chorioidea near the area postrema, and were studied in detail by electron microscopy. The cytological features of the cyst cell and its junctional relationship to neighboring cells imply that cyst cells are derived from ependymal and choroid epithelial cells. The cyst cells usually contact directly the perivascular spaces of postremal, choroidal or pial capillaries, where the cytoplasm is often considerably attenuated. The cystic lumen is commonly filled with a flocculent material. The limiting membrane of the cystic lumen, which frequently bears cilia and microvilli, has the same thickness as the surface cell membrane. In many cases, the cyst is surrounded by the cytoplasm of a single cell. In some cases, however, two cells participate in the formation of the cyst, although one is only a slender process and joined by a zonula occludens with the main cyst cell. Horseradish peroxidase (HRP) injected into the cerebrospinal fluid (CSF) space failed to enter the cystic lumen. A possible significance of the cyst in relation to the CSF and blood circulation was considered.     

Cholecystokinin-octapeptide like immunoreactivity in the area postrema of the rat and cat

The distribution of cholecystokinin-8 (CCK-8)-like immunoreactivity in the area postrema of the rat and cat was visualized using the peroxidase, antiperoxidase technique. In the rat the greatest amount of immunostaining occurred in peripheral regions of the area postrema at intermediate and rostral levels. Caudally, scattered immunoreactivity predominated. After colchicine treatment, numerous immunoreactive somata were observed throughout the area postrema. The cat area postrema had a different and more complex pattern of immunostaining than the rat. Moderate to dense accumulations of immunostaining occurred in the ventromedial region of the area postrema bordering the solitary tract and dorsal vagal nuclei. The central region of the area postrema possessed scattered amounts of immunoreactivity at rostral levels. Following colchicine treatment, no visible CCK-8-like immunoreactive cell bodies were observed in the cat area postrema. Results of the present investigation provide morphological evidence for the role of CCK-8 in cardiovascular regulation and satiety. The difference in the distribution of CCK-8 in the rat and cat suggest a possible role in the emetic reflex.     

The microvascular pattern and perivascular linings of the area postrema

Summary The microvasculature and perivascular linings of the area postrema (A.P.) were studied electron microscopically with the ultrathin section and freeze-etching techniques. Special attention was given to the intercellular contacts of the different cellular entities. Two types of microvascular segments were identified. The endothelium of these vascular segments reveals fenestrations and a high pinocytotic activity. There are no significant differences in the frequency and distribution of the endothelial openings between both types of capillaries. The endothelium of the blood vessels, however, is joined by different types of tight junctions. Focal tight junctions occur between pericytes and the endothelium, and between leptomeningeal cellular elements in the perivascular space. The cell membrane of the perivascular glia shows intramembrane particles which are either distributed at random or organized in the form of membrane-associated orthogonal particle complexes (MOPC, Dermietzel, 1974). The significance of these findings is discussed with respect to the modified blood-brain barrier mechanism in the A.P.Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 114 Bionach) to R. DermietzelThe authors are indebted to Mrs. D. Schünke and Mr. R. Eichner for technical assistance and Mr. A. Stapper for preparing the diagram     

The functional and structural borders between the CSF-and blood-dominated milieus in the choroid plexuses and the area postrema of the rat

Summary In the borderline area between the hemal milieu of the choroid plexuses (PC) and the interstitial cerebrospinal-fluid (CSF) compartment, ground substances displaying increased amounts of basal lamina-like material and containing negatively charged sulfated glycosaminoglycans appear to be endowed with selective properties. They may function as a sieve or filtration barrier gradually controlling the passage of substances between the two milieus, depending on their charge and molecular weight. Special structural features and functional properties of ependymal cells are associated with such bordering structures. These ependymal cells are transitional elements between choroid epithelium and ciliated ependymal cells. As judged from experiments with horseradish peroxidase and conventional electron microscopy, occluding junctions at the basal pole of these cells prevent a rapid alteration in the milieu conditions, enabling gradual change from hemal to CSF composition near the bases of these transitional ependymal cells. The borderline structures between the hemal milieu of the PC and the area postrema (1) are established by leptomeningeal cells which face a hemal mileu, (2) are endowed with conspicuous tight junctions, and (3) produce a flocculent substance, the light-microscopic equivalent of which is PAS positive. These structures probably establish an effective barrier between the two milieus of different composition. The functional characteristics and the morphology of the meningeal cells facing the hemal milieu of neurohemal regions resemble closely the neurothelial cells, which are interposed between the CSF milieu and the hemal milieu in the dura mater. The present results suggest that the location between the hemal and the CSF milieu is decisive for the transformation of leptomeningeal cells into neurothelial elements.Supported by the Deutsche Forschungsgemeinschaft (Grant Kr 569/5-1)Dedicated to Professor Dr. med., Dr. med. vet. h.c., Dr. phil. h.c. Andreas Oksche on the occasion of his 60th birthday     

Cells capable of uptake of horseradish peroxidase in some circumventricular organs of the cat and rat

Summary The structure of mesenchymal cells distributed in some of the hypendymal organs of the circumventricular system in the cat and rat was demonstrated after intravenous injection of high doses of horseradish peroxidase. These cellular elements were observed in the vicinity of blood vessels of the organon vasculosum laminae terminalis, subfornical organ and area postrema. Electron-microscopically, these cells located between the basal laminae of the brain parenchyma and the blood capillaries show long cellular processes encircling fenestrated capillaries. Light and electron-microscopic examination revealed that this cell type is identical with the horseradish peroxidase-uptake cells, previously reported in the vicinity of the hypophysial portal system. Such phagocytic cells may be considered as a cellular component intervening between the brain parenchyma and the blood stream, playing a role in selective barrier functions in the above-mentioned circumventricular organs where a blood-brain barrier in the classical sense of the definition is lacking.This work was supported by grant No. 437002 from the Ministry of Education, Science and Culture, Japan     

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat

Summary Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2–3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.     

Patch-clamp study on membrane properties and transmitter activated currents of rabbit area postrema neurons

Using the patch-clamp technique in combination with sliced tissue preparation the membrane properties of newborn rabbit area postrema neurons were investigated. The neurons responded upon depolarization with a fast Na ⁺-current followed by an inactivating and non-inactivating K ⁺-current. GABA-activated currents were investigated resulting in a large Cl^--conductance, indicating the expression of GABA_A-receptors. The expression of glutamate receptor mRNA was studied by in situ hybridization and electrophysiological measurements of these receptors by means of the patch-clamp technique. As a main result it was found that ionotropic glutamate receptors in the area postrema are composed of flop variants of the GluA-, GluB- and GluC-subunits.Abbreviations AP area postrema - GABA -aminobutyric acid - Glu glutamate - I–V current-voltage - SFO subfornical organ     

Fine structure of the ependymal cells in the area postrema of the domestic fowl

Summary The ultrastructure of the ependymal cells in the area postrema of the domestic fowl was studied by scanning and transmission electron microscopy. The ependymal surface of the area postrema is covered with many furrows and ridges. These ridges consist of ependymal cells aggregated in a fan-like shape. The ependymal cell lacks clustered cilia, microvilli are few, and a long basal process extends through the parenchymal layer of the area postrema. Within the cytoplasm as well as in the basal process, a spherical body with a diameter ranging from 1.5 to 2 gmm is occasionally observed.This work was supported by a Scientific Research Grant, No. 144017, from the Ministry of Education of Japan to Professor M. YasudaThe authors are grateful to Drs. T. Fujioka and T. Watanabe for their valuable advice     

The penetration of sodium into the brain following a cisternal injection of sodium chloride with particular emphasis on the area postrema

Summary Following an intracisternal injection of sodium chloride, sodium has been localized in paraventricular and subpial tissues of the posterior fossa by means of the pyroantimonate histochemical technique, with the use of a buffered pyroantimonate medium. The electron dense deposit is present in these tissues within 4 minutes after injection and is found only extracellularly except in the area postrema. This finding supports the contention that sodium is chiefly an extracellular ion and that the cerebrospinal fluid and the extracellular fluid are in equilibrium at these sites. In the area postrema, an intracellular precipitate is noted in the vesicular structures of the atypical astrocytes of this structure and in pinocytic vesicles of the large blood vessels. The intraglial localization of sodium in the area postrema is discussed in relation to a possible function of this structure as a regulator of cerebrospinal fluid ionic content.This work was supported by grant number NB-08549-02 from the National Institute of Neurological Disease and Stroke, Bethesda, Maryland.     

Characterization of orexin A immunoreactivity in the rat area postrema

The distribution of orexin A immunoreactivity and the synaptic relationships of orexin A-positive neurons in the rat area postrema were studied using both light and electron microscopy techniques. At the light microscope level, numerous orexin A-like immunoreactive fibers were found within the area postrema. Using electron microscopy, immunoreactivity within fibers was confined primarily to the axon terminals, most of which contained dense-cored vesicles. Both axo-somatic and axo-dendritic synapses made by orexin A-like immunoreactive axon terminals were found, with these synapses being both symmetric and asymmetric in form. Orexin A-like immunoreactive axon terminals could be found presynaptic to two different immunonegative profiles including the perikarya and dendrites. Occasionally, some orexin A-like immunoreactive profiles, most likely to be dendrites, could be seen receiving synaptic inputs from immunonegative or immunopositive axon terminals. The present results suggest that the physiological function of orexin A in the area postrema depends on synaptic relationships with other immunopositive and immunonegative neurons, with the action of orexin A mediated via a self-modulation feedback mechanism.     

Immunohistochemical localization of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat

Summary The indirect peroxidase-antiperoxidase immunohistochemical technique was used to investigate the possible presence of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat. Considerable numbers of VIP-immunoreactive fibers were seen in the pineal gland. A moderate amount of VIP-immunoreactive fibers was present in the median eminence, the posterior lobe of the pituitary and the area postrema, but only few fibers were found in the organum vasculosum laminae terminalis. No immunoreactivity was observed in the subfornical organ or the subcommissural organ. The circumventricular organs investigated were completely free of VIP-immunoreactive perikarya. In the circumventricular organs, VIP-immunoreactive fibers were visible between the parenchymal cells and in the perivascular spaces. The presence of coarse VIP-immunoreactive terminals in apposition to the portal vessels in the external layer of the median eminence indicates that VIP may be secreted directly into the pituitary portal circulation, thus influencing the anterior pituitary cells. The presence of large VIP-immunoreactive boutons in the posterior lobe of the pituitary suggests a secretion of VIP directly into the systemic circulation. In the pineal gland, a dense innervation by VIP-immunoreactive fibers was found in the peripheral superficial part of organ, with fibers penetrating into its central portion where they mainly terminate near in vicinity of the capillaries. In the area postrema, VIP-immunoreactive material was mainly found at the ventral border of the organ. In addition to the secretion of VIP into the bloodstream via the circumventricular organs, this study provides evidence that VIP exerts specific influence on the cellular elements of these organs.     

家兔延髓最后区对心血管活动的调节

目的:探讨家兔延髓最后区(AP)对心血管活动的调节作用及机制.方法:以10%脲酯和1%氯醛糖混合静脉麻醉家兔,人工呼吸.给AP不同频率(10 Hz～80 Hz)电刺激,观察平均动脉压(MAP)和心率(HR)的变化;及损毁CVLM或RVLM后AP兴奋的心血管效应改变.结果:电刺激AP,低频(10 Hz、20 Hz)刺激使MAP下降、HR减慢;高频(60 Hz、80 Hz)刺激使MAP升高,HR减慢.损毁CVLM后刺激AP,20 Hz刺激引起的降压作用消失(P＜0.01),80 Hz刺激效应同前(P＞0.05).损毁RVLM后刺激AP,20 Hz、80 Hz刺激引起的降压、升压作用均消失(P＜0.01).结论:低频电刺激AP,引起降压和心率减慢,高频电刺激AP引起升压和心率减慢;前者可能与兴奋CVLM有关,不同电刺激引起的心血管效应均通过RVLM介导.     

Distribution of anti-Leu-7, anti-Leu-11a and anti-Leu-M1 immunoreactivity in the brain of the adult rat

Summary This study reports a specific cross-reactivity of the three anti-human-hematopoetic-cell monoclonal antibodies, anti-Leu-7 (HNK-1), anti-Leu-11a (NKP-15), and anti-Leu-M1 (MMA), with different epitopes in the brain of the adult rat. The distribution of these epitopes in rat brain is determined by means of immunohistochemistry in paraffin-embedded frontal serial sections.The reaction pattern of anti-Leu-11a monoclonal antibody is very similar to that of polyclonal antibodies against the myelin basic protein. Both antisera give a specific reaction with myelinated fibers. Immunoreaction products with the anti-Leu-7 monoclonal antibody are found as diffuse, mostly punctiform material in the neuropil and even more evident as small granules coating the cell surface of many neurons. In the white matter anti-Leu-7 reveals a moderate reactivity, which occurs predominantly as spots and fine-stranded material within the myelinated fiber tracts.Anti-Leu-M1 immunoreactivity is present between myelinated fiber bundles of the white matter, where it has a reticulate appearance, and as fine-granulated material within the grey matter of the cortex and the nuclei. The characteristic feature in the grey matter is that of irregularly shaped immunopositive plaques, which are often located around small blood vessels. The cytoplasm of glial and neuronal cells appeared negative with this MAB.The exact topographical distribution of the Leu-7 and Leu-M1 epitopes throughout the rat brain is described. The present hypotheses concerning the nature of this shared antigenicity between hematopoetic cells and nervous tissue are discussed.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Distribution patterns of neurotensin-like immunoreactive cells in the gastro-intestinal tract of higher vertebrates

Summary The endocrine system of the gastro-intestinal tract of selected species representing the five higher vertebrate classes was investigated with reference to occurrence and distribution of neurotensin-like immunoreactive cells. Using antibodies against C-terminal and N-terminal fragments of neurotensin and against the C-terminal sequence of xenopsin it was demonstrated that the intestine of all species studied contains endocrine, neurotensin-like immunoreactive cells. However, large differences in localization and frequency of these neurotensin-like immunoreactive cells were found. Except for a teleostean fish, neurotensin-like immunoreactive cells in the gastro-intestinal tract were more frequent in non-mammalian vertebrates than in mammals. In contrast to mammals, where the highest density of neurotensin-like immunoreactive cells was present in the ileal mucosa, in the non-mammalian vertebrates studied the corresponding cells were most abundant in the pyloric-duodenal junction. The exact mapping of neurotensin-like immunoreactive cells is presented throughout the entire gastro-intestinal tract of six species (Rattus, Coturnix, Lacerta, Rana, Xenopus, Carassius) including a quantitative evaluation of sequential serial sections.     

Histochemical localization of FMRFamide-like immunoreactivity in the rat brain

Molluscan cardioexcitatory neuropeptide or FMRFamide is present in the invertebrate central nervous system (CNS) and FMRFamide like peptide has been demonstrated in the mammalian CNS. In this study, the distribution of FMRFamide immunoreactivity was studied in rat brain using the indirect immunofluorescent method. The highest number of FMRFamide staining cell bodies was found in the nucleus (n) arcuatus. N. paraventricularis, n. hypothalamus, n. ventromedialis, n. dorsomedialis and n. tractus solitarii also contained high numbers. FMRFamide positive nerve fibers and terminals were widely distributed. The septal complex contained high densities, especially in n. interstitialis striae terminalis. N. paraventricularis hypothalami, n. paraventricularis, n. hypothalamicus, n. ventromedialis and n. dorsomedialis showed a high to very high degree of immunoreactivity. In myelencephalon, n. tractus solitarii had the densest innervation. Spinal cord had a dense band of FMRFamide positive fibers in lamina I and II of the dorsal horn. The present findings support a neurotransmitter role for a FMRFamide like peptide in the mammalian brain, possibly related to endocrine and autonomic regulation as well as pain modulation.     

Glucagon-like immunoreactivity in hypothalamic neurons of the rat

Summary Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.     

Distribution and immunochemical character of neurotensin-like material in representative vertebrates and invertebrates: Apparent conservation of the COOH-terminal region during evolution

Using a radioimmunoassay for bovine neurotensin (NT) and various region specific antisera which react selectively with different portions of the molecule, the presence of immunoreactive NT (iNT) in a wide range of vertebrate and invertebrate species has been demonstrated. While antisera directed towards the N-terminal region of NT recognized only mammalian forms of NT, antisera directed towards the C-terminal region of NT recognized materials from all species examined, including representatives of all vertebrates and invertebrate classes. When extracts of the brain and gut of the vertebrates examined were chromatographed on Sephadex G-25 multiple NT-like substances were observed, and the patterns of iNT obtained seemed to fall into three groups: (a) mammals, (b) birds and reptiles and (c) amphibians and fish. Extracts of invertebrates also exhibited multiple peaks of iNT on Sephadex G-25 and the profiles observed resembled those for lower vertebrates. Partially purified iNT obtained from chicken, turtle, dogfish and lobster was shown to increase hematocrit and induce cyanosis in anesthetized rats. These findings indicate (a) that NT-like substances appear to be present throughout the animal kingdom, (b) that the C-terminal region of NT is highly conserved while the N-terminal region varies, and (c) that in any one animal multiple substances sharing C-terminal homologies with NT exist. These findings are consistent with the notion that NT and related peptides participate in important processes basic to animal life and that their functioning depends highly upon elements located in their C-terminal regions. They further suggest the existence of an entire family of NT-related peptides in each animal form, possibly distributed differently and functioning differently in the various organs of the animal.     

Gastrin releasing peptides increase Fos-like immunoreactivity in the enteric nervous system and the dorsal vagal complex

We and others have shown that gastrin-releasing peptide (GRP) reduces food intake. In this study, we determined the activation of the gastrointestinal and dorsal vagal complex (DVC) neurons by various forms of GRP to determine the pathway involved in this reduction. We found the following: (1) GRP-10, -27 and -29 (2.1 nmol/kg, i.p.) increased the Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of the stomach and the area postrema (AP) of the DVC; (2) GRP-27 and GRP-29 increased the Fos-LI in the myenteric plexus of the duodenum; and (3) only GRP-29 increased the Fos-LI in the submucosal plexus of the duodenum. In conclusion, GRP may reduce food intake by activating the area postrema. The enteric neurons may have a potential role in this reduction through the direct activation of the AP or exerting local gut actions, such as the stimulation of gut motility or secretions.     

Neurokinin A-like immunoreactivity in feline dental pulp: its distribution,origin and coexistence with substance P-like immunoreactivity

Summary The distribution and origin of neurokinin A (NKA)-like immunoreactivity were investigated in feline dental pulp by an indirect immunofluorescence method. NKA-containing nerve fibres with varicosities, which entered the dental pulp via apical foramen, were distributed throughout this tissue. Many NKA-containing nerve fibres were localized around blood vessels, but some were observed apart therefrom. At the odontoblastic layer, thin NKA-containing nerve fibres were observed running straight toward the pulp-predentinal border between odontoblasts. After inferior alveolar nerve section, all NKA-containing nerve fibres disappeared in the dental pulp, while the removal of the superior cervial ganglion resulted in no change in the distribution of these fibres. The correlation of NKA-like immunoreactivity and substance P (SP)-like immunoreactivity was also investigated by double-immunofluorescence technique. The distribution of NKA-containing nerve fibres was very similar to that of SP-containing nerve fibres; it appeared that all NKA-containing nerve fibres contained SP.