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PubMed1978

Serotonin nerve terminals in the locus coeruleus of adult rat: a radioautographic study.

Serotonin (5-HT) nerve terminals in the locus coeruleus (LC) of adult rat were visualized by high-resolution radioautography, in order to examine their distribution, fine structural features and intimate relationships with norepinephrine neurons. In animals pretreated with a monoamine oxidase inhibitor, prolonged intraventricular perfusion of 10(-4) M [3H]5-HT resulted in a specific identification of most if not all 5-HT axonal varicosities in LC. These terminals were equally distributed between the dorsal and ventral divisions of the nucleus. Their density was approximated at 10(7) per cu.mm within the middle third of the LC. In electron microscope radioautographs, the labeled 5-HT varicosities averaged 0.9 micron in diameter. They all exhibited a distinctive storage organelle, in the form of microvesicles and microcanaliculi (15-25 nm in diameter) partly filled with electron-dense material and usually aggregated in association with several large dense-core vesicles. While this finding of intrinsic morphological characteristics appeared compatible with a special cellular origin or regional differentiation, it was also suggestive of particular functional properties and/or mode of action. In a sample comprised of some 500 sectional profiles from labeled 5-HT varicosities in LC, a small proportion only (less than 10%) exhibited morphologically defined synaptic junctions. These rare contacts were invariably made with dendritic processes and never observed on the noradrenergic perikarya. It is therefore concluded that, in the LC, non-synaptic as well as synaptic mechanisms might be involved in the modulation and transneuronal regulation of norepinephrine neurons by 5-HT afferents.

Animals↗

PubMed1979

Three-dimensional organization of the endoplasmic reticulum in supraoptic neurons of the rat. A structural functional correlation.

Double impregnation staining of tissue was used to study on thick sections the three-dimensional configuration of the peripheral endoplasmic reticulum in neurosecretory neurons of the supraoptic nucleus in control and water-deprived rats. According to the spatial organization of the endoplasmic reticulum, two types of neurons are described in this report: Type I neurons which predominated in control rats (70%) exhibited characteristically large lamellar structures connected to loosely anastomosed tubules. In type II neurons, which prevailed in water-deprived rats (85%) the endoplasmic reticulum had the appearance of a highly-developed network of interconnected tubules, with no lamellar structures. Double impregnation staining combined with high resolution radioautography after [3H]leucine administration showed that the tubular configuration of the endoplasmic reticulum was the main active site of protein synthesis by contrast with the lamellar components, whose activity seemed poor. In terms of protein synthesis, the three-dimensional configuration of the peripheral endoplasmic reticulum of the supraoptic neurons appeared therefore to be closely connected with their functional state.

Animals↗

PubMed1979

The effects of acute morphine treatment on the incorporation of [3H]L-lysine by normal and regenerating facial nucleus neurons.

Although opiates significantly alter RNA and protein synthesis in a variety of neuronal cell types, their effect on the biosynthetic activity of regenerating neurons has not been investigated. In the present study, the effect of morphine on the incorporation of [3H]L-lysine into proteins of facial nucleus neurons was examined by light microscopic radioautography. Silver grains present within various compartments of normal and regenerating (3-, 7-, 14- and 21 days post-axotomy) neurons from saline-treated Wistar rats were compared with the amount present in similar cells from animals receiving 40 mg/kg morphine sulfate i.v. At 14- and 21-days post-axotomy, regenerating neurons were larger and the grain count in the emulsion over these cells was greater than that observed in normal (unoperated) neurons. In normal facial neurons, the accumulation of lysine into the nucleus and nucleolus was significantly lower 60 min after morphine administration. However, morphine's inhibition of lysine incorporation was even more pronounced in regenerating neurons. In these cells, nuclear lysine uptake was depressed at 3 and 7 days, while maximum inhibition of cytoplasmic incorporation occurred at 14-days post-axotomy. Morphine administration decreased nucleolar lysine incorporation at all survival intervals.

Animals↗

PubMed1979

Radioautographic characterization of a serotonin-accumulating nerve cell group in adult rat hypothalamus.

Intensely labeled nerve cell bodies were identified by radioautography within the pars ventralis of nucleus dorsomedialis hypothalami (hdv), following intraventricular perfusion with 10(-5) or 10(-4) M tritiated serotonin [3H]5-HT in adult rats pretreated with a monoamine oxidase inhibitor. This selective reaction, which involved approximately 1000 neurons on each side of the third ventricle, was unaltered by concomitant administration of 10(-3) M non-radioactive norepinephrine, and was absent after intraventricular injection of 10(-5) or 10(-4) M tritiated norepinephrine. The 3H-labeled 5-HT nerve cell bodies were loosely grouped within the inner and caudal half of the hdv, and appeared morphologically similar to the unreactive neurons among which they were interspersed. Within the same region, numerous labeled axonal varicosities were also detected, which were never found in synaptic contact with the reactive cells. If the 3H-labeled 5-HT neurons contain endogenous 5-HT, they might constitute an intrinsic source of 5-HT innervation in the adult rat hypothalamus.

Animals↗

PubMed1979

The filum terminale of the frog spinal cord, a non transformed preparation: I. Morphology and uptake of gamma-aminobutyric acid.

The filum terminale of the frog spinal cord is a rather pure glial cell preparation, largely devoid of neuronal elements. gamma-Aminobutyric acid (GABA) is taken up by the frog filum terminale (FT) via a Na+-dependent, ouabain-inhibited, saturable high affinity transport system with a Km of 2.7 x 10(5) M. The rate of the FT GABA uptake is significantly greater than the velocities observed in the spinal cord. In fact, the Vmax increases caudally beyond the level of the last root, and is maximal in the FT per se. beta-Alanine is a competitive inhibitor of the FT high affinity transport system for GABA (Ki 11.1 x 10(-5) M). In addition to GABA, the FT also takes up beta-alanine, glycine, glutamate and aspartate at rates significantly higher than those shown by the spinal cord of the frog. Light and electron microscope level radioautography clearly shows that GABA uptake occurs primarily in the glial cells and also in ependymal cells present in the FT. In that the FT contains few ependymal cells and a large number of glia, it is fair to state that most of the GABA accumulated by the FT reflects the glial transport of this amino acid. Unlike the adult frog, the spinal cord of the tadpole does not show any regional differences in the rate of GABA transport during early development. However, during later developmental stages, the rates of GABA transport increase in the caudal portion of the tadpole cord as compared to the more rostral areas. Close to metamorphosis, the terminal portion of the tadpole cord, which is destined to become the filum terminals of the frog, accumulates GABA at rates not greatly different from those observed in the FT of the adult frog. Therefore, the tadpole spinal cord is a useful preparation in which to study the dynamic properties of normal non-transformed glia as influenced by a changing neuronal population, whereas the frog FT is a unique preparation for the study of some properties of normal glia largely in the absence of neurons.

Amino Acids↗

PubMed1979

Innervation of hippocampal explants by central catecholaminergic neurons in co-cultured fetal mouse brain stem explants.

The ability of central catecholaminergic neurons to grow into and establish functional connections with the hippocampus in vitro was studied using organotypic tissue culture. Brain stem explanted from the region of the locus coeruleus and hippocampal explants, from 18-day fetal mice, were maintained as co-cultures and were also grown separately. After 1-4 weeks these tissues were analyzed by glyoxylic acid-induced histofluorescence, by light and electron microscopic radioautography after incubation with [3H]norepinephrine, and by electrophysiology. Brain stem explants exhibited specifically fluorescent catecholaminergic cell bodies and varicose fibers after 2-4 weeks in culture. In contrast, no fluorescent cells or neurites could be seen in isolated hippocampal cultures grown for 2-3 weeks in vitro. When hippocampal explants were grown near brain stem explants, catecholaminergic fibers grew out of the brain stem and entered the hippocampus. In additional experiments, co-cultures of brain stem and hippocampus were incubated with [3H]norepinephrine (0.5 micron) and the monoamine oxidase inhibitor nialamide (100 micron). Radioautographic analyses revealed that brain stem neurites which entered the hippocampus took up norepinephrine, whereas neurites in the isolated hippocampal explants did not. Electron microscopic studies of the hippocampus showed varicose axon terminals within the hippocampus to be preferentially labeled. Although close relationships could be seen between labeled axons and dendrites, junctions exhibiting the membranous modifications associated with synapses were never seen. Electrophysiological studies suggested that the catecholaminergic neurites within the hippocampus were functional. Complex synaptically mediated slow wave discharges could be evoked by electrical stimuli in isolated hippocampal explants. Introduction of the beta adrenergic antagonist propranolol (0.4-4.3 micron) did not alter, or slightly depressed, these hippocampal discharges. On the other hand, in hippocampus-brain stem co-cultures, these concentrations of propranolol enhanced the complex hippocampal responses to brain stem or hippocampal stimuli. Similar enhancement of hippocampal responses by propranolol also occurred in these cocultures after acute surgical extirpation of the brain stem explant. The data suggest, therefore, that the action of propranol was probably to block adrenergic inhibitory connections with hippocampal synaptic networks. These experiments provide morphological and electrophysiological evidence that catecholaminergic neurons from fetal mouse brain stem maintained in organotypic tissue culture can grow into and functionally innervate the hippocampus.

Animals↗

PubMed1979

Release of [3H]serotonin and its binding protein from enteric neurons.

The release of [3H]5-HT and its binding protein, SBP, from the guinea pig enteric nervous system was analyzed. Release of both [3H]5-HT and [3H]NE from strips of longitudinal muscle with adherent myenteric plexus preloaded with the respective radioactive amine was evoked by high K+ and the ionophore X537A. However, Ca2+-dependence could not be shown for [3H]5-HT release by either agent or for [3H]NE release by X537A. However, Ca2+-dependence (as well as inhibition of release by high Mg2+ and tetrodotoxin) could be demonstrated for the release of radioactivity evoked by electrical field stimulation of everted segments of ileum preloaded by perfusion through the serosal lumen with [3H]5-HT. Light and electron microscopic radioautography revealed that the sources of released radioactivity were axons, especially axonal varicosities containing a mixture of small clear and large dense-cored vesicles. SPB, but not the cytosol marker protein, lactic dehydrogenase, was spontaneously released from the perfused everted ileum. A marked increase in SBP (but not LDH) release was provoked by electrical field stimulation at 10 Hz, and this increased release (but not the spontaneous release) was Ca2+-dependent. It is concluded that SBP and 5-HT are probably stored together, at least in part in vesicles, and that both can be released by exocytosis from depolarized axon terminals.

Animals↗

PubMed1980

An analysis of the efferent connections of the septal area in the cat.

The neuroanatomical organization of the efferent connections of the septal area in the cat was analyzed by the use of anterograde ([3H]leucine radioautography) and retrograde (horseradish peroxidase histochemistry) tracing techniques. The results indicate that the lateral septal nucleus projects to the nuclei of the diagonal band, preoptic area, lateral hypothalamus, and supramammillary region. The projections of the septofimbrial nucleus supply the nuclei of the diagonal band and the medial habenular nucleus. Projection targets of the vertical limb of the diagonal band are widespread and include the preoptic area, lateral hypothalamus, anterior limbic cortex, amygdala, medial habenular nucleus, interpeduncular nucleus and hippocampal formation. The projection from the vertical limb to the hippocampal formation is organized in a topographical manner in such a fashion that cells positioned near the midline project to the dorsal hippocampus and adjoining subicular cortex while fibers originating from cells situated more laterally project to more ventral parts of the hippocampal formation. In general, the projections from the horizontal limb were similar to those from the vertical limb, but several differences were noted. Fibers arising from the horizontal limb are distributed to the ventral tegmental area and interpeduncular nucleus but this region seems to lack a projection to either the habenular complex or to the ventral aspect of the hippocampal formation. Fibers arising from the bed nucleus of the anterior commissure are distributed to the preoptic region, lateral hypothalamus, supramammillary region, posterior aspect of the medial mammillary nucleus and lateral habenular nucleus.

Animals↗

PubMed1980

[Identification of the vestibular projections in the oculomotor nuclei in the cat by autoradiography and electron microscopy].

The projection of vestibular pathways to the oculomotor nuclei ws investigated by electron microscopic radioautography. Unilateral injection of tritiated amino acids into the rostral vestibular complex was used in order to characterize the location and to identify the different types of labeled synaptic terminals involved in these pathways. In the normal oculomotor nuclei, 4 types of synaptic boutons were identified. Following the labeling of the vestibular synapses, in the ipsilateral oculomotor nucleus, types I and II boutons are the most prominent group and make up 75% of the synaptic vesicles, they are distributed on the cellular soma and the large dendrites of the oculomotor neurons. In contrast, in the contralateral oculomotor nucleus, type III boutons which are smaller and have larger diameter synaptic vesicles were predominant; they are prevalent on the distal part of the dendritic tree. From the results obtained, a relationship between the present anatomical findings and previously published physiological studies is established. The following conclusion is suggested: the inhibitory vestibular inputs probably terminate on the oculomotor neurons by these large types I and II boutons and the excitatory vestibular inputs by the smaller type III boutons. Also discussed is the complexity of the pattern of afferentation and the functional arrangement of the oculomotor nuclei.

Animals↗

PubMed1980

Retrograde axonal transport following injection of [3H]-serotonin into the olfactory bulb. II. Radioautographic study.

Radioautography was used to study the intraneuronal distribution of [3H]-serotonin (5-HT) and/or its derivatives selectively taken up by the olfactory bulb (OB) serotonergic terminals and subsequently transported to their parent cell bodies in the midbrain raphe nuclei. This was done 24 h after injection of [3H]5-HT into the main OB of rats either pretreated or not with monoamine oxidase (MAO) inhibitor. A prior mechanical obstruction of the rostral ventricular cavities prevented diffusion of the tracer towards cerebrospinal fluid. Heavily labelled nerve cell bodies were found mainly in the ipsilateral raphe dorsalis nucleus (RDN) and to a lesser extent in the raphe centralis nucleus. The radioautographic reaction often extended to dendritic processes while sparing the nucleus. A diffuse reaction was also observed but limited to the raphe area. The supraependymal 5-HT fibers were found to be free of labelling. Neither local destruction of catecholaminergic terminals with 6-OHDA, nor absence of MAO inhibition, impaired this radioautographic pattern, while destruction of serotonergic terminals with 5,6-dihydroxytryptamine in OB resulted in the disappearance of labelled axonal varicosities and neurons in the OB and the RDN respectively. At the electron microscopy level, labelled cell bodies in the RDN were medium-sized (12-15 micrometers). Silver grains were localized mainly on mitochondria and, to a lesser extent, on lysosomes and endoplasmic reticulum but spared the nucleus and the nucleolus. Silver grains were also found near the nuclear membrane and outside the neuronal membrane. The observation of heavy metal impregnated thick sections confirmed the preferential localization of silver grains on mitochondria with or without inhibition of MAO. These results could account for the subcellular compartments involved in the retrograde axonal transport of [3H]5-HT and its subsequent degradation and/or dendritic release.

Animals↗

PubMed1981

A projection from the entorhinal cortex to the nucleus accumbens in the rat.

A study was performed in which both anterograde ([3H]leucine radioautography) and retrograde (horseradish peroxidase (HRP) histochemistry) tracing methods were employed to identify the origin of the fimbrial projection to the nucleus accumbens in the rat. The data reveal that this pathway arises predominantly from layers II--III of the anterior two-thirds of entorhinal cortex rather than from any part of hippocampal formation.

Animals↗

PubMed1981

Contribution of axonal transport to the renewal of myelin phospholipids in peripheral nerves. I. Quantitative radioautographic study.

Kinetics of phospholipid constituents transferred from the axon to the myelin sheath were studied in the oculomotor nerve (OMN) and the ciliary ganglion (CG) of chicken. Axons of the OMN were loaded with transported phospholipids after an intracerebral injection of [2-3H]glycerol or [3H]labeled choline. Quantitative electron microscope radioautography revealed that labeled lipids were transported in the axons mainly associated with the smooth endoplasmic reticulum. Simultaneously, the labeling of the myelin sheath was found in the Schmidt-Lanterman clefts and the inner myelin layers. The outer Schwann cell cytoplasm and the outer myelin layers contained some label with [methyl-3H]choline, but virtually none with [2-3H]glycerol. With time the radioactive lipids were redistributed throughout and along the whole myelin sheath. Since [2-3H]glycerol incorporated into phospholipids is practically not re-utilized, the occurrence of label in myelin results from a translocation of entire phospholipid molecules and from their preferential insertion into Schmidt-Lanterman clefts. In this way, the axon-myelin transfer of phospholipid contributes rapidly to the renewal of a limited pool of phospholipids in the inner myelin layers. When [methyl-3H]choline was used as precursor of phospholipids, the rapid appearance of the label in the inner myelin layers was interpreted also as an axon-myelin transfer of labeled phospholipids. However, the additional labeling of the outer Schwann cell cytoplasm adjacent to Schmidt-Lanterman clefts and of the outer myelin layers reflects a local re-incorporation of the base released from the axon. By these two processes, the axon contributes to purvey the inner myelin layers with new phospholipids and the Schwann cells with new choline molecules.

Animals↗

PubMed1981

Optic fiber segregation in goldfish with two eyes innervating one tectal lobe.

An ipsilateral retinotectal projection was induced by ablating one tectal lobe. Radioautography indicated that the ipsilateral that the ipsilateral projection initially spread out continuously over the remaining tectal lobe. With time, the continuous projection progressively changed into a rostrocaudally oriented banded projection that was comprised of high and low silver grain density bands. The undamaged native projection from the contralateral eye also became transformed into high and low density bands. The results indicate that foreign fibers displace portions of the native projection. Complete segregation of the two projections was not found at any time point examined. Low density bands did not represent spillover of label from a high density band since cobalt-filled optic fibers were found in low density bands. Quantitative analysis indicated that the contralateral projection occupied more tectal area than the ipsilateral projection. Area occupied by a projection, band width and band frequency showed appreciable between-fish variability. Correlations between band width and per cent of area occupied by a projection approached unity, indicating that large projections were isolated with wider bands. A model is proposed to account for induced banding in lower vertebrates.

Animals↗

PubMed1981

Synaptic remodeling of serotonin axon terminals in rat agranular cerebellum.

In order to assess the influence of the target zone on the synaptic modeling of central serotonin (5-HT) axons, the 5-HT innervation of the posterior vermal cortex was studied by high resolution radioautography in both normal and X-ray-induced agranular rat cerebella, following topical application of [3H]5-HT. Two major systems of 5-HT afferents were identified in normal cerebellar cortex: (1) typical mossy fibers confined to the granular layer and (2) fine beaded axons diffusely distributed through all layers. The density of this innervation was estimated to be approximately 240,000 varicosities/cu.mm of cortex. The labeled mossy terminals all established synaptic contacts with the dendrites of granule cells. In contrast, only 3% of the varicosities belonging to the 'diffuse system' exhibited active zones in single thin sections, implying that less than 9% were actually engaged in junctional synaptic relationships. In the agranular cerebellar cortex, all 5-HT terminals belonging to the so-called 'diffuse system'. Their density was more than 8 times higher than in normal rat (2 million/cu.mm of cortex), an increase accounted for by the smaller volume of the experimental cerebellum. Thirty-five per cent of these 5-HT varicosities were seen in synaptic contact, indicating that all established at least one junctional complex. Most of these synapses were made on the branchlet spines of Purkinje cell dendrites, but some were also observed on the dendritic shafts of Golgi cells. Thus, in the absence of granule cells, the 5-HT innervation of rat cerebellar cortex evolves from a mostly 'non-junctional' into an entirely 'junctional' input. This finding indicates that the territory of innervation can exert a determinant influence on the synaptic modeling of incoming 5-HT afferents.

Afferent Pathways↗

PubMed1981

Distribution of membrane glycoproteins among the organelles of a single identified neuron of Aplysia. I. Association of a [3H]glycoprotein with vesicles.

[3H]N-acetylgalactosamine injected into the cell body of R2, the giant cholinergic neuron in the abdominal ganglion, is rapidly incorporated into membrane glycoprotein and glycolipid. Incorporation, which is localized to the injected cells, occurs at a constant rate for approximately 15 h. By that time, 83% of the labeled macromolecules are associated with membranes. Quantitative electron microscopic radioautography of the cell body shows that labeling of membranous organelles is selective: the Golgi apparatus, endoplasmic reticulum, and lucent and compound vesicles are labeled, while the nucleus, end-stage lysosomes, and mitochondria are not. SDS-polyacrylamide gel electrophoresis of the total membranes from more than 40 R2s examined individually resolves reproducibly 5 major labeled glycoprotein components. In order to determine which of these area associated with vesicles, we isolated a labeled vesicle fraction from R2 using a combination of differential centrifugation and filtration on a column of glass beads with 200 nm pores. This fraction was consistently enriched in [3H]glycoproteins I (180,000 Daltons) and V (90,000 Daltons) relative to those fractions containing larger organelles. These experiments suggest that different organelles contain characteristic membrane components.

Acetylgalactosamine↗

PubMed1981

Distribution of membrane glycoproteins among the organelles of a single identified neuron of Aplysia. II. Isolation and characterization of a glycoprotein associated with vesicles.

Glycoprotein-I (mol.wt. 180,000) is associated with a vesicle fraction from the cytoplasm of R2, the giant cholinergic neuron in the abdominal ganglion. Electron microscopy has shown that R2 contains both lucent and compound vesicles. We have used anisomycin, an inhibitor of protein synthesis, to provide evidence that Component-I is a constituent of somatic compound vesicles. In the presence of the drug, [3H]N-acetylgalactosamine injected into R2 is incorporated almost exclusively into Component-I. Quantitative electron microscopic radioautography of treated cells shows a marked increase in the proportion of silver grains over compound vesicles and a decrease in labeling of other organelles compared with untreated cells. Analysis of Component-I, isolated using the chaotropic agent lithium diiodosalicylate, shows it to contain fucose, N-acetylgalactosamine, and N-acetylglucosamine. Proteolytic digestion with pronase yields a complex pattern of glycopeptides. The proportion of [3H]N-acetyl-galactosamine in these glycopeptides is altered in the presence of anisomycin. These results together with radioautographic analyses suggest that large carbohydrate chains are elaborated within the endoplasmic reticulum and that smaller chains are added in the Golgi region or on the membrane of the compound vesicle.

Acetylgalactosamine↗

PubMed1981

'Acrylamide-induced' neuropathy and impairment of axonal transport of proteins. II. Abnormal accumulations of smooth endoplasmic reticulum as sites of focal retention of fast transported proteins. Electron microscope radioautographic study.

The distribution of fast axonally transported proteins was studied by electron microscope radioautography in ciliary ganglia of chickens treated or not treated with acrylamide. At 3 h after the intracerebral injection of [3H]lysine, the preganglionic axons of the untreated chickens displayed few silver grains, mainly associated with smooth endoplasmic reticulum (SER) profiles. In most axons of acrylamide-treated chickens, a similar pattern was observed, except in axons which exhibited focal and intense labeling underneath the axolemma: clusters of silver grains indeed overlayed peripheral accumulations of tubulovesicular profiles of SER, dense core vesicles and mitochondria. After impregnation with heavy metals, electron microscope observation of 1 micrometer thick sections showed a locally disorganized SER forming a complex network of tubules intermingled with vesicles and mitochondria. Such a local disorganization of the peripheral SER in the distal part of the axons, could be responsible for the focal stasis of fast transported proteins; it seems to be one of the earliest changes detectable in axons damaged by acrylamide treatment.

Acrylamides↗

PubMed1981

Protein and RNA synthesis in cerebral microvessels: a radioautographic study.

The distribution of protein and ribonucleic acid (RNA) synthesized by telencephalon slices was determined by radioautography at the light microscope level. Previous studies had shown that, in telencephalon slices, the cells associated with the cerebral microvasculature were synthesizing protein at very high rates compared to other cerebral cells such as neurons. In this study, therefore, particular emphasis was placed on the distribution of newly synthesized protein and RNA among the various cell types of the microvasculature. Rat telencephalon slices were incubated with either [3H] leucine or [3H] uridine for 1 h. The 3H-protein that was found in association with microvessels was present in endothelial cells, pericytes and smooth muscles cells. There was also extensive labeling of cellular processes adjacent to the abluminal side of the basement membrane and extending as far as 15 micrometer from the lumen. The latter processes may have been astrocyte glial projections. All [3H] RNA found associated with microvessels was, however, restricted to endothelial cells and pericytes. This data is discussed in relation to the induction of a 71,000 dalton protein which occurs within minutes of the preparation of tissue slices, and requires the synthesis of new RNA.

Animals↗