Topographical differences of RNA labelling in rat brain after intraventricular administration of labelled RNA precursors

Summary ¹⁴C-uridine or ¹⁴C-orotic acid was injected into the third or fourth brain ventricle of adult rats. The rate of incorporation of these precursors into the RNA of various brain regions was studied by autoradiography. 0.5 hour, 1 hour, 2 hours and 4 hours after the injection of labelled uridine and/or orotic acid into the third ventricle, a very uneven labelling of different brain regions was observed. The highest grain density was found over the ventricular walls and in the closely adjacent brain tissue; the intensity of labelling decreased sharply with distance from the ventricular lumen. 24 hours after intraventricular injection, a medio-lateral gradient of grain density was no longer observed. An intense labelling of leptomeninx (especially at the base of the brain) and of ependymal cells was observed at all time intervals investigated. At time intervals 0.5–2 hours the grain density of these structures surpassed by a considerable amount the grain density over neurons, glial cells or neuropile.Two hours after the injection of ¹⁴C-orotic acid into the fourth ventricle, the grains were mainly localised over leptomeningeal cells and vessels at the base of the brain and in the adjacent narrow strip of brain tissue. The rest of the brain was only very faintly labelled.     

A Golgi study on the cerebrospinal fluid (CSF)-contacting neurons in the paraventricular nucleus of the Pekin duck

The present investigation based on classical neurohistological techniques (Nissl-staining, Golgi-impregnation) was focussed on the cytoarchitecture of the periventricular layer of the paraventricular nucleus in the Pekin duck. This region is endowed with intraependymal neurons, the perikarya of which are mostly covered by a thin ependymal lamella. Several of the intraependymal neurons were shown to give rise to dendrites extending into the third ventricle. An additional population of nerve cells located in the deeper layers of the periventricular region also gained direct access to the cerebrospinal fluid by means of long dendrites terminating with a bulbous-like swelling in the third ventricle. This cerebrospinal fluid (CSF)-contacting dendrite branched off several times in a rectangular fashion to give rise to collaterals running in the subependymal or periventricular layers. The axons of these CSF-contacting neurons were followed into the magnocellular portion of the paraventricular nucleus. Small bipolar nerve cells with processes parallel to the surface of the third ventricle occupied a subependymal position. The isodendritic magnocellular neurons of the paraventricular nucleus emitted dendritic processes that reached the basal pole of the ependymal cells. The complex arrangement of the periventricular layer of the paraventricular nucleus might provide the structural basis for the mechanisms of cerebral osmoreception defined by means of physiological parameters.     

Distribution of 1-14C palmitic acid in brain tissue after intraventricular injection in the conscious cat]

The distribution of (1-14C) palmitic acid in the brain tissue following the injection into the cerebral ventricles of conscious cats was investigated. The radioactive material was found in the brain tissue surrounding the cerebral ventricles and in the cerebral cortex, but in varying amounts : the smallest amounts were found in the cerebral cortex, while the highest in the thalamus and in the hippocampus. Radioactive material was also found in the peripheral venous blood. The amount of the radioactive material in the grey matter lining the cerebral ventricles as well as in the cerebral cortex was time-dependant. The labelled material in the structures surrounding the cerebral ventricles and in the cerebral cortex increased within first four hours after its intraventricular administration. Thereafter, throughout subsequent 48 hours either it slowly disappeared in the caudate nucleus and in the thalamus, or it was retained in the hypothalamus and in the floor of the IV ventricle.     

Ultrastructural localization of radiolabelled L-dopa in the endocrine hypothalamus of the rat

Summary Light and electron microscopic autoradiography has been employed to define the neuroanatomical patterns of uptake and binding of radiolabelled L-dopa in the endocrine hypothalamus of the rat. A dorsomedial continuum of arcuate and periventricular neurons selectively sequester ³H L-dopa 20 min following its intraventricular infusion. By 40 and 60 min following the infusion labelling of neurons is minimal and supports the notion of rapid degradation. Other cell compartments such as tanycytes demonstrate uptake of ³H L-dopa. The ultrastructural localization and distribution of radiolabelled L-dopa (or its metabolites) in the rodent hypothalamus is discussed with respect to mechanisms and cell compartments involved in neuroendocrine regulatory processes.Supported by USPHS Program Project Grant NS-11642-04 (DES) and RR-05403Career Development Awardee RO4GM-70001     

An immunocytochemical and ultrastructural study of a specialized glial region of the medulla oblongata of the adult and juvenile grey mullet

Electron microscopy together with glial fibrillary acidic protein (GFAP) and vimentin immunocytochemistry reveals the presence of a specialized glial region in the octavolateral area of the medulla oblongata of adult and juvenile grey mullets, Chelon labrosus. Glial cells, which can he characterized as ependymal and subependymal cells, originate in the walls of the lateral recess of the fourth ventricle. They contain numerous gliofilaments and are connected through frequent gap junctions. Electron microscopy also reveals the lack of nerve cell parikarya and processes in this region. Immunocytochemistry reveals that the glial cells are strongly GFAP-positive in both adults and juveniles, whereas vimentin is only detected in this region in juveniles. The meninges associated with this glial region contains connective fibres, formed of very thick collagen fibres, that arc metachromatic and PAS-positive under light microscopy. These findings strongly support a structural role for this medullary specialization. Differences between adults and juveniles in the distribution of GFAP- and vimentin-immunoreactive structures in other regions of the medulla oblongata are also reported.     

Systemic site of action for pressor effect of angiotensin II injected into the fourth cerebral ventricle of rats?

Angiotensin II (ANG II) causes a systemic pressor effect when injected into the cerebral ventricles. In the rat fourth ventricle, the effective doses for the ANG II pressor effect are over 100 times larger than in the systemic circulation. Considering the discrepancy of doses, the possibility that ANG II may reach the systemic circulation and promote pressor effects, following injection into the fourth ventricle, was investigated. The effects on blood pressure of different vasoactive peptides that produce pressor responses when injected into the central nervous system were compared. Dose-response curves were obtained for intravenous or fourth cerebroventricular injections of ANG II, lysyl-vasopressin (LVP), bradykinin (BK), or endothelin-1 (ET-1). The ED50 ratios for intracerebroventricular/intraveneous injections were 110 for ANG II, 109 for LVP, 0.01 for BK, and approximately 0.4 for ET-1. In cross-circulation preparations, pressor responses occurred in the donor rat following injection into the fourth cerebral ventricle of the recipient animal, showing that effective doses of ANG II, administered to the fourth cerebral, reach the systemic circulation. The same results were obtained for the microinjection of 4 nmol of LVP into the fourth cerebral ventricle of recipient animals. High-performance reverse-phase liquid chromatography analyses of arterial blood showed that approximately 1% of the [125I]ANG II injected into the fourth cerebral ventricle may be recovered from the systemic circulation a few seconds after the microinjection. The systemic administration of the ANG II receptor antagonist losartan blocked the response to ANG II injected into the fourth ventricle whereas antagonist administration in the same ventricle did not. Angiotensin injections into the lateral ventricle produced pressor responses that were reduced by antagonist administration to the same ventricle but not by systemic administration of the antagonist. The data suggest that the pressor effect resulting from ANG II or LVP injections into the fourth cerebral ventricle may be due to the action of this peptide in the systemic circulation. On the other hand, the pressor effect due to ANG II microinjection into the lateral ventricle apparently results from the direct stimulation of central periventricular structures.     

Ependyma and supraependymal structures in some areas of the fourth ventricle in the rat

The ependyma was investigated in five areas of the rat ventricle system by means of both light and electron microscopy. The columnar, cuboidal and flattened types of the ependymal cells were mainly seen. All of them were seen in the fourth ventricle, while in the aqueductus cerebri and in the central canal the flattened type of the cell was lacking. An unusual variation as to the form of the ependymal cells was found on the roof of the fourth ventricle. Three groups of intraventricular structures were found in all investigated parts of the ventricle system: supraependymal globular structures containing irregularly arranged cristae, supraependymal protrusions appearing as homogeneous contents, and nerve profiles including nerve endings and nerve axons. The morphological characteristics of the ependyma and intraventricular profiles in the fourth ventricle allow to suppose a certain role of these structures in the exchange of various materials between the CSF, ependyma and neuropile.     

Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads.

When mouse spermatozoa were briefly exposed in culture to radioactively labelled DNA (pSV2CAT plasmid), radioactivity could be detected by high-resolution autoradiography on the surface and within the nucleus of the spermatozoa. Spermatozoa from other mammalian species (boar, bull, man) could also bind foreign DNA. With the exclusion of human spermatozoa, which in most experiments showed very low labelling values, labelling percentages (evaluated by light microscope autoradiography) ranged between 39 and 78%. In all four species the DNA-binding ability was mainly confined to a specific region of the sperm head (equatorial segment and postacrosomal region), and the sperm-DNA association kinetics were rapid (maximum values were reached within 20-40 min). The data also indicate that factor(s) in seminal plasma might protect spermatozoa from accidental transfection by foreign DNA that may be present in the genital tracts from bacterial or viral sources.     

Incorporation of labelled amino acids into the acoustic cortex of the cat

Labelled amino acids, 14C-leucine, 3H-glycine, 14C-GABA, 14C-glutamic acid and 14C-aspartic acid were applied to the surface of the cat's auditory cortex in dried filter paper strips for 40 min. The distribution of the amino acids taken up by the cortex was examined by autoradiography.The right acoustic area was stimulated by 2 cps acoustic clicks, through an ear-phone. Leucine showed cellular localization which was considerably dispersed by cortical excitation. Glycine was concentrated in nerve cells, mainly in layers I-II, but excitation intensified and extended its incorporation. GABA was accumulated rather diffusely in layers I-II with scattered cellular labelling which was depressed by stimulation. Glutamic and aspartic acids did not show any characteristic distribution pattern. The authors emphasize that the incorporation of glycine seems to be a good indicator of neurons being in excitation. The localizations of GABA uptake shows close correlation with the site of its physiological action.     

Fate map of serotonin transporter-expressing cells in developing mouse heart

Serotonin regulates cardiovascular functions during embryogenesis and adulthood. However, the source of serotonin in the cardiovascular system and the role of circulating serotonin and serotonin transporter (SERT) in the regulation of cardiovascular functions are still unclear. We used a cell fate approach to map the regions of the mouse heart expressing SERT, utilizing a Cre/loxP system driven by SERT gene expression. Cell labelling was first detected at E10.5 and was mapped until E18.5. We found labelling in the outflow tract, part of right ventricle and to a very limited extent in the left ventricle. Interestingly, the distribution pattern of SERT-fated cells was remarkably similar to that obtained with markers of the second heart field lineage. In addition, we observed staining of atrioventricular valves, consistent with valvular abnormalities observed in SERT-/-animals. Overall, our data reveal specific and regionally restricted distribution of SERT-expressing cells in the developing heart of mouse.     

A rhesus monkey model for continuous infusion of drugs into cerebrospinal fluid

A new rhesus monkey model with two intraventricular catheter systems was developed to examine the pharmacokinetics and neurotoxicity of chemotherapeutic agents administered by continuous intraventricular infusion. A lateral ventricular catheter system implanted in the lateral ventricle and attached to a subcutaneous access port on the animal's back is used for infusion of drugs into the ventricle. A Pudenz catheter implanted in the fourth ventricle and connected to a subcutaneous Ommaya reservoir permits repetitive CSF sampling in unanesthetized animals. The model was evaluated in five animals for over 12 months for catheter patency, surgical complications, and utility in studying the pharmacokinetics of continuous intraventricular infusion of methotrexate. There were no perioperative complications. Three of the five monkeys maintained both systems successfully. The other two animals developed staphylococcal ventriculitis, one at 7 days as a result of manipulation of the incision by the animal leading to cellulitis around the catheter site and subsequent ventriculitis, the other at 5 months. Both animals were treated successfully with antibiotics and catheter removal. An infusion of 0.05 mg of methotrexate over 24 hours maintained ventricular drug concentrations of 1 mol/L without evidence of neurotoxicity. This new model has applications both for the development of continuous intraventricular infusion as a therapeutic approach for the treatment of meningeal cancers in humans and as a research tool to study the distribution and elimination of drugs from the CSF.     

A golgi study on tanycytes and liquor-contacting cells in the posterior hypothalamus of the newt

Summary Golgi methods were employed to study neurons and ependymal tanycytes in the posterior hypothalamus of the newt. The tanycytes send a few coarse, spiny or barbed processes towards the pia mater. In the periventricular grey, the neurohistological methods show common neurons, ranging from a multipolar to a plumed organization, and abundant liquor-contacting cells. These cells, possibly neurons, give rise to a process that reaches the cerebro-spinal fluid, and terminates in a spindle-shaped swelling, with a thin thread at its tip. In other cells, the intraventricular endings are bulbous or finger-like. The occurrence of: (1) branches of the liquor-contacting process, running parallel to the infundibular surface; (2) infundibular processes which end at the base or between the ependymal cell bodies; and (3) axons coursing in the same position, all indicates that the subependymal layer is a site for complex intercellular relationships. The significance of liquor-contacting cells and tanycytes is discussed, in view of the possibility that they may represent part of a system for hypothalamic regulation in response to changes in the CSF.     

Steroid hormone target cells in the periventricular brain: relationship to peptide hormone producing cells.

Steroid hormone concentrating cells in hypothalamic and extrahypothalamic regions are reviewed and the topographic relationship to the periventricular brain and the ventricular recess organs is discussed. Steroid hormone target cells in the brain are considered feedback sites and production sites of polypeptide hormones. The anatomical distribution of estrogen, androgen and progestin target neurons, as defined by autoradiography, is compared with the localization of antibodies to luteinizing hormone-releasing hormone and somatostatin in perikarya of neurons, as characterized by immunocytochemistry. Around the optic recess of the third ventricle in the lamina terminalis and the preoptic nucleus as well as in the periventricular nucleus of the hypothalamus, the steroid hormone target neurons and the assumed polypeptide hormone producing neurons occupy corresponding sites.     

Does ex vivo labelling of proliferating cells in colonic and vaginal mucosa reflect the S-phase fraction in vivo?

The ex vivo labelling of DNA-synthesizing epithelial cells in colonic and vaginal mucosa was compared with in vivo labelling. For this purpose, in vivo S-phase cells were labelled with [3H]thymidine (Tdr) and ex vivo labelling was continued by culturing tissue specimens in bromodeoxyuridine (BrdU). Various methods of tissue culture were employed in order to improve diffusion of medium (and BrdU) in the tissue. BrdU and 3H-TdR labelling were evaluated by immunohistochemistry and autoradiography respectively. Ex vivo labelling resulted in a patchy distribution of labelled cells, which did not correspond with the 3H-TdR labelling pattern obtained in vivo. Under the described conditions ex vivo labelling does not appear to be a reliable for estimation of the proliferative activities in vivo.     

The origin of the dendritic reticulum cell

Summary ¹⁴C-5,6-DHT-Melanin was injected into the left lateral ventricle of adult rats and its fate followed by light and EM autoradiography and by TEM of structures identified as labeled in preceding light micrographs. Shortly after injection, melanin particles were seen ingested by supraependymal and epiplexus cells, by cells residing in the pia-arachnoid, i.e. free subarachnoidal cells and perivascular cells, and by subependymally located microglia-like cells with intraventricular processes. Up to day four, an increase in the number of labelled phagocytes in the CSF was noted which transformed into typical reactive macrophages. After this time, many intraventricular melanin-laden phagocytes formed rounded clusters; cells of such clusters were subsequently found to invade the brain parenchyma by penetrating the ependymal lining and to accumulate in the perivascular space of brain vessels. ¹⁴C-Melanin-storing macrophages were found in the marginal sinus of the deep jugular lymph nodes suggesting emigration of CNS-derived phagocytes via lymphatics or prelymphatics that contact the subarachnoidal space compartment. This does not exclude the possibility that some of the macrophages leave the brain via the systemic circulation by penetrating the vascular endothelium; these may be disposed of in peripheral organs other than the lymph nodes.The ability of supraependymal, epiplexus, free subarachnoidal and perivascular cells in the pia and of subependymal microglia cells to accumulate synthetic melanin by phagocytosis suggests that these cells are local variants of the same type of resting potential phagocytes of the mammalian brain. The present study shows that ¹⁴C-5,6-DHT-melanin is an ideal phagocytic stimulant and marker for phagocytosis.Supported by grants from the Deutsche Forschungsgemeinschaft     

The paraventricular organ of mormyrid fish: uptake or release of intraventricular biogenic amines?

The paraventricular organ of Gnathonemus petersii was investigated with light and electronmicroscopical techniques. It contains high concentrations of dopamine, noradrenaline and serotonin, but the synthesizing enzymes are not or hardly present. Consequently, the cerebrospinal fluid-contacting neurons might pick up their biogenic amines from the ventricular fluid. Dense subependymal axonal plexuses in the everted telencephalon probably release these substances into the ventricle. However, electronmicroscopical observations suggest release rather than uptake by the paraventricular organ. The possible significance of intraventricular release, transport and uptake of biogenic amines is discussed.     

ACTH acts via an anterior ventral third ventricular site to elicit grooming behavior

Intracerebroventricular but not parenteral application of ACTH has been shown to elicit excessive grooming behavior in rats and mice. This behavior is elicited by administration of ACTH into the lateral, third, or fourth ventricles. Plugging of the cerebral aqueduct with cold cream fails to prevent grooming in response to lateral ventricle injection of ACTH. However, cold cream plugs in the third ventricle can prevent the subsequent induction of grooming behavior by lateral ventricle injection of ACTH, but only when the plugs are located in the anterior ventral third ventricle in the region of the organum vasculosum laminae terminalis (OVLT) and median eminence. These data suggest the anterior ventral third ventricle as the periventricular site of action of ACTH in eliciting excessive grooming, although it is possible that peptides taken up in this area are transported to other regions to elicit the behavioral response.     

Rearrangement of serotonin-immunoreactive fibers in the denervated rat suprachiasmatic nucleus after transplantation of fetal raphe tissue

Summary Pieces of fetal midbrain raphe tissue were transplanted into the third ventricle or the ventral hypothalamic region near the suprachiasmatic nucleus (SCN) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. The ability of grafted serotonin neurons to reinnervate the SCN in the host rats was studied by means of immunohistochemistry 1 and 3 months after transplantation. In both the intraventricular and intraparenchymal transplant experiments, reinnervation by outgrowing serotonin fibers was observed in the hypothalamus of host rats at 1 and 3 months after surgery. At both survival periods, there was no abundant arborization of serotonin fibers in the SCN, while the preoptic and periventricular areas of the host rats displayed a pattern of serotonergic innervation resembling that in normal (untreated) rats. It is suggested that within the SCN the regenerating serotonin fibers may be exposed to an inhibitory environment.     

Cell-surface radioiodination with the sparingly soluble catalyst Iodogen. Difference between the dividing and non-dividing populations of rodent thymocytes.

The surface proteins of dividing and non-dividing subpopulations of rat and mouse thymocytes have been labelled by using a new method of radioiodination. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography of the labelled proteins shows distinct differences in labelling between the mouse and rat cells and also, in the case of the rat, between the dividing and non-dividing populations.