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PubMed1979

The efferent connections of the nucleus raphe centralis superior in the rat as revealed by radioautography.

By chronically implanting a glass micropipette filled with tritiated leucine in the raphe centralis superior of the rat, the projection of this nucleus was traced by radioautography. The majority of the ascending projections were located within the ventral tegmental area and, further rostrally, the median forebrain bundle. Along the course of this bundle numerous fibers branched successively into the mammillary peduncle, the fasciculus retroflexus, the stria medullaris, the fornix and the cingulum. The most significant projections included the ones to the interpeduncular nucleus, the mammillary bodies, the habenular nuclei and the hippocampus. No projections were detected in the striatum, the cortex piriformis or the amygdala. Descending projections diffused to the pontine reticular formation and central gray through the medial and the dorsal longitudinal bundles. In addition widespread projections were also seen in nuclei located near the raphe centralis superior: raphe nuclei, dorsal and ventral tegmental nuclei.

Animals↗
PubMed1981

'Acrylamide-induced' neuropathy and impairment of axonal transport of proteins. I. Multifocal retention of fast transported proteins at the periphery of axons as revealed by light microscope radioautography.

The axonal transport of proteins was studied in ciliary ganglia of control and acrylamide-treated chickens. After an intracerebral injection of [3H]lysine, the distribution of axonally transported proteins was analyzed in the preterminal segments of preganglionic axons and in their caliciform nerve endings by means of quantitative light microscope radioautography. By 7 days after the injection of [3H]lysine, the concentration of labeled proteins in the preganglionic axons was either similar or slightly increased in acrylamide-treated chickens as compared to controls. In contrast, by 3 h, whereas the axons of controls were poorly radioactive, 20-30% of the axons in acrylamide-treated chickens displayed focal and intense labeling at their periphery; simultaneously, in acrylamide treated chickens, 20-30% of the nerve endings contained a decreased amount of radioactive proteins. It is concluded that acrylamide induces multifocal retention of fast axonally transported proteins in preterminal segments of certain axons.

Acrylamides↗
PubMed1984

Regional differences in binding of [3H]LSD and [3H]5-HT in calf hippocampal slices revealed by radioautography and rapid filtration studies.

Previous radioautographic experiments demonstrated that binding sites labeled by [3H]5-HT and [3H]LSD in rat brain were seen in all layers of CA1, CA4 and the dentate gyrus but not in fields CA2 and CA3 of the hippocampus. In an attempt to confirm this observation we performed binding assays on homogenates from selected areas of calf hippocampus since the small size of the rat hippocampus precluded using preparations from this animal for this purpose. Studies on homogenates from calf hippocampal regions, were done after we determined that the binding to slices in vitro was similar in the calf and rat. Binding of both [3H]5-HT and [3H]LSD by homogenates of CA1 and dentate gyrus, but not of CA3, was saturable. These studies show that the qualitative differences in binding site distribution within the calf hippocampus seen by radioautography reflect quantitative differences in the densities of binding sites revealed by the homogenate studies.

Animals↗
PubMed1985

The noradrenergic innervation of vasopressin neurons in the paraventricular nucleus of the hypothalamus: an ultrastructural study using radioautography and immunocytochemistry.

Immunocytochemical and radioautographic procedures were combined at the ultrastructural level to study the noradrenergic synaptic input to vasopressin neurons in selected portions of the paraventricular nucleus of the hypothalamus (PVN) of the rat. Radioactive norepinephrine (NE) was infused into the lateral ventricle or applied topically to the region of the PVN. After appropriate survival times, brain tissues were processed for ultrastructural immunocytochemical demonstration of vasopressin using a monoclonal antibody. [3H]NE varicosities were detected by electron microscopic radioautography. In the periventricular zone of the PVN, radioactive varicosities were numerous accounting for 20-30% of all nerve terminals in this zones. These NE terminals primarily innervated dendritic processes of non-vasopressinergic neurons. Although an occasional axosomatic synapse was observed, input to vasopressin positive neurons was exclusively to their dendrites. In the lateral magnocellular sub-nucleus of the PVN (designed pvl2), noradrenergic terminals were fewer in number accounting for only 1-2% of the total. These terminals were found predominately but not exclusively making axodendritic synapses onto non-vasopressin processes. In both regions, many of the radiolabeled terminals had well-defined membrane appositions with their post-synaptic partners which included a synaptic cleft and post-synaptic density of varying thickness. In both the periventricular zone and the lateral magnocellular regions, noradrenergic varicosities were seen in close proximity to numerous blood vessels.

Animals↗
PubMed1985

Localization and characterization of brain somatostatin receptors as studied with somatostatin-14 and somatostatin-28 receptor radioautography.

The localization and characterization of receptors for somatostatin-14 (S-14) and somatostatin-28 (S-28) were studied in the rat brain using the iodinated agonists [Tyr0,D-Trp8]S-14 and [Leu8,D-Trp22,Tyr25]S-28 as tracers. Slide-mounted frozen sections were used for the radioautographic localization and biochemical characterization of somatostatin receptors. In the latter case counting was performed on scraped off serial sections from rostral regions of the brain. Specificity studies demonstrated that either tracer could be displaced with S-28, S-14 or their agonists. The N-terminus fragment (1-12) of S-28 as well as a number of unrelated peptides were unable to compete with either tracer, indicating that the binding capacity for ligand-receptor recognition is located in the C-terminal portion of S-28. Scatchard analysis of saturation curves gave a one-site interaction with Kd values of 0.42 +/- 0.09 nM and 0.32 +/- 0.04 nM for the S-14 and S-28 iodinated agonists, respectively. By radioautography, the distribution of receptors for both S-14 and S-28 appeared very similar with high levels of binding in the deep layers of the cortex, the cingulate cortex, the claustrum, the locus coeruleus and most structures of the limbic system. Treatment with cysteamine, which caused a somatostatin depletion in the brain, was required to observe labeling in the hypothalamus. In some caudal areas of the brain, especially in the cerebellar nuclei, the solitary tract nucleus and the nucleus of the vagus nerve, only labeling with the S-28 agonist could be detected. This S-28 binding could be displaced by native S-14 (10(-6) M). Generally, there was a correlation between the localization of somatostatin receptors and that of immunoreactive somatostatin, as evaluated by immunocytochemistry. However, in some areas, an inverse correlation between receptor and peptide concentrations was observed. These results are in agreement with previous data suggesting that somatostatin could act as a neurotransmitter or neuromodulator in several brain areas.

Animals↗
PubMed1987

Quantitative analysis of glucose after transient ischemia in the gerbil hippocampus by light and electron microscope radioautography.

Changes in glucose uptake in the gerbil hippocampus were studied by high-resolution [3H]2-deoxyglucose radioautography under sham and postischemic conditions. Sections of dorsal hippocampi were fixed by chemical fixatives or rapid-freezing and freeze-substitution techniques. Light and electron microscope radioautograms showed that the cell soma of each CA1 neuron subjected to transient ischemia revealed various degrees of glucose uptake. In the neuropil of the CA1 stratum radiatum, glucose uptake was higher in the thin dendrites of the ischemic group. Cell damage due to transsynaptic stimulation is suggested by these results.

Animals↗
PubMed1980

Characterization and radioautography of [3H]LSD binding by rat brain slices in vitro: the effect of 5-hydroxytryptamine.

Binding of D-[3H]lysergic acid diethylamide (LSD) to rat coronal brain slices and its blockade by 5-hydroxytryptamine (5-HT) had characteristics similar to those of brain homogenates in respect of KD, kinetics and reversibility of binding. Radioautography was done on slices that had been incubated in 6 nM [3H] LSD and on adjacent slices incubated in the same concentration of tritiated LSD plus 10(-5) M of 5-HT. Choroid plexus showed densest labeling of [3H] LSD. In neuropil, dense labeling occurred within parts of the hippocampal formation except for fields CA2 and CA3 which were sparsely labeled. All layers of the cortex except the posterior cingulate gyrus were labeled by LSD. 5-HT blocked labeling of choroid plexus, hippocampal formation, septum, pons, medulla and parts of cortex but only reduced labeling of most other structures. LSD binding sites may relate to some of its pharmacological effects.

Animals↗
PubMed1991

Lysosomal cobalamin accumulation in fibroblasts from a patient with an inborn error of cobalamin metabolism (cblF complementation group): visualization by electron microscope radioautography.

Cobalamin (Cbl, vitamin B12) bound to transcobalamin II (TCII) enters cultured fibroblasts by receptor-mediated endocytosis. Following degradation of the TCII, Cbl is subsequently found in either the cytoplasm bound to methionine synthase or in the mitochondria bound to methylmalonyl CoA mutase. In fibroblasts from patients belonging to the cblF complementation group, Cbl is found free in the cell and is not transferred to the above two target enzymes. Quantitative EM radioautography was utilized to visualize intracellular Cbl in fibroblasts from cblF patients and from normal subjects. In cblF cells, 60% of all silver grains were assigned to lysosomes, with only 12.6% over cytoplasm and 1.2% over mitochondria. In contrast, in control cells, only 4.7% were assigned to lysosomes, with 47% to cytoplasm and 23.4% to mitochondria. Subcellular fractionation showed that in cblF cells, the majority of label was associated with clearly recognizable lysosomes. These studies conclusively demonstrate that secondary lysosomes accumulate Cbl in cblF disease.

Autoradiography↗
PubMed1991

Characterization of pericellular [125I]Tyr0 DTrp8 somatostatin binding sites in the rat arcuate nucleus by a newly developed method: quantitative high-resolution light microscopic radioautography.

In the present work we characterized the kinetic properties of [125I]somatostatin pericellular binding sites in the arcuate nucleus of the hypothalamus of the rat by quantitative high-resolution light microscopic radioautography. In order to determine whether these pericellular binding sites corresponded to functional receptors, their properties were compared with those of previously well-characterized [125I]somatostatin binding sites present on neuronal processes on the same sections in the stratum radiatum of the CA1 of the hippocampus. Radiolabelled sections were analysed by densitometry using a Biocom image analysis system coupled with a Leitz orthoplan microscope. The linear relationship between optical densities and radioactive standards allowed us to quantitate [125I]somatostatin-specific binding. Binding was time- and temperature-dependent, and saturable and specific in the arcuate nucleus as in the CA1 of the hippocampus. Saturation experiments indicated a single receptor population of binding sites with KD values of 0.2 +/- 0.1 nM in the arcuate nucleus and 0.6 +/- 0.4 nM in the CA1. In both structures, displacement curves obtained with somatostatin 14 and somatostatin 28 were monophasic, but shallow, while the somatostatin analogue SMS 201-995 induced a biphasic displacement, suggesting two populations of binding sites. In both regions binding was GTP-dependent. Desaturation procedures (in vivo by cysteamine and in vitro by preincubating with GTP) resulted in an increase in the number of measurable binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry.

The neuronal distribution of cannabinoid receptor in the adult rat brain is reported, combining receptor binding radioautography using the synthetic psychoactive cannabinoid ligand CP55,940 with in situ hybridization histochemistry using oligonucleotide probes complementary to rat cannabinoid receptor cDNA. In the cerebral cortex, especially in the frontal and cingulate cortex, dense binding was found in layers I and VI together with slight mRNA levels in a majority of both pyramidal and non-pyramidal-shaped neurons and of high mRNA levels in a moderate number of non-pyramidal-shaped neurons especially in layers II-III and V-VI. In the hippocampal dentate gyrus, very dense staining was found in the molecular layer together with high mRNA levels in a moderate number of hilar neurons close to the granular layer. In Ammon's horn, especially in the CA3 sector, very dense binding was found in the dendritic layers together with slight mRNA levels in the majority of the pyramidal cells and high mRNA levels in a moderate number of interneurons. In the basal ganglia, binding was very dense in the lateral putamen, substantia nigra pars reticulata, globus pallidus and entopeduncular nucleus, moderate in the medial putamen and caudate; and slight in the accumbens, together with slight to moderate mRNA levels in the striatal medium-sized neurons. Together with slight binding, slight to moderate mRNA levels were found in the majority of the neurons in the subthalamic nucleus. No binding and mRNA were found in the substantia nigra pars compacta and ventral tegmental area. Slight to moderate binding was found together with slight to moderate mRNA levels in the majority of neurons in the anterior olfactory nucleus; septum, especially medial septum and diagonal band of Broca; amygdala, especially basolateral amygdala; lateral habenula; ventromedial hypothalamic nucleus; lateral interpeduncular nucleus; central gray, dorsal cochlear nucleus; parabrachial nucleus; dorsal pontine tegmentum; pontine nuclei; commissural part of the nucleus tractus solitarius; inferior olive and dorsal horn of the spinal cord. In the cerebellum, very dense binding was found in the molecular layer together with slight mRNA levels in the majority of the granule cells and moderate mRNA levels in the basket and stellate cells. In conclusion, this study provides, for the first time, indirect assessment of the neurons containing cannabinoid receptor in the entire adult rat brain and will serve as a basis for future direct morphological confirmation using receptor immunohistochemistry and for functional studies.

Animals↗
PubMed1990

S phase duration measurement by combined PCNA/cyclin immunostaining and radioautography after a single pulse-labelling with 3H-thymidine.

A method for measuring S phase duration is described and evaluated that combines single pulse labelling with 3H-thymidine (TdR), detected by radioautography, and proliferating cell nuclear antigen (PCNA)/cyclin immunostaining to replace the second pulse labelling of the classical double-labelling method. Conditions were set up in which nuclei showing one or both types of label were readily distinguished, hence allowing to verify that cell fluxes in and out of S phase were equal. S phase durations thus measured in different tissues of the mouse were concordant with those obtained by the double 3H-TdR labelling or from labelled mitoses curves. Our method might be used with archived samples of methanol-fixed cells or tissues, singly labelled with 3H-TdR or with bromodeoxyuridine.

Animals↗
PubMed1995

Experimental thyrotoxic myopathy: radioautography of protein synthesis in skeletal muscle and motor neurons of spinal cord.

The total protein synthesis was studied in skeletal muscle and spinal motor neurons in experimental thyrotoxic myopathy (TM) mice by using radioautography with 3H-methionine. A significant increase of mean specific radioactivity was found in motor neurons of mice with TM. No statistical differences were established in the level of 3H-methionine inclusions into muscle fibre proteins of experimental and control animals. Thus, the total protein synthesis in motor neurons is significantly increased with TM, while there is no change in the skeletal muscle. Our data suggest that muscular weakness in TM is not the consequence of protein metabolism disorders in skeletal muscle fibres.

Animals↗
PubMed1983

Synthesis and assembly of connecting-piece proteins as revealed by radioautography.

The synthesis and assembly of connecting-piece proteins have been studied during spermiogenesis in the rat by electron microscopy and radioautography following intratesticular injection of radiolabeled amino acids [3H]proline and [3H]cystine. Early in spermiogenesis (steps 1-7) the two centrioles that give rise to the connecting piece are essentially unmodified. During the 6.5-day period between steps 8 and 15, the major elements of the connecting piece (striated columns and capitulum) gradually become assembled from an electron-dense material that is deposited around the walls of the centrioles; throughout this period, protein molecules containing proline and cystine are synthesized by the step 8-15 spermatids and incorporated into the developing neck region. These proteins subsequently become permanent structural components of the connecting piece. Following completion of the major elements in step 15, few additional proteins are added to the connecting piece during the final steps 16-19 of spermiogenesis.

Animals↗
PubMed1987

Localization of newly synthesized protein precursors of basement membrane in the embryonic central nervous system as revealed by radioautography.

Using light- and electron microscope radioautography, the dynamics of newly synthesized protein precursors taking part in elaboration of brain basement membrane have been examined. The data presented give evidence that all cell types (endothelial, pericytal, and astroglial cells) surrounding the brain basement membrane contribute to its composition. A quantitative uptake analysis of 3H-proline indicates a progressive decline in the amount of labeled precursor in the examined cell types with a corresponding increase in deposition of the label at the presumptive basement membrane. The endothelial cells plays the main role in the membrane elaboration followed by pericyte and astrocyte which participate at lesser but equal degree in this synthesis.

Animals↗
PubMed1985

Observations on the distribution and movement of cadmium in epithelial cells of rat small intestine by light- and electron-microscope radioautography.

The localization of cadmium in the absorptive cells of the small intestine has been studied by light- and electron-microscope radioautography at intervals in the first 27 h after intubation of radiocadmium into the stomach of rats. Light-microscope autoradiographic studies showed that cadmium was taken up very quickly by the absorptive cells, and radioactivity in the absorptive layer was disseminated over the entire area of the absorptive cells. Electron-microscope autoradiography showed that there was no concentration of radioactive cadmium in any particular site in the epithelial cells or in the lamina propria. It suggests that the transport mechanism of cadmium through the intestine may be nonspecific. Radioautographic investigation using radiocadmium may provide useful information for cadmium distribution and movement in rat small intestine.

Animals↗
PubMed1966

Synthesis of the carbohydrate of mucus in the golgi complex as shown by electron microscope radioautography of goblet cells from rats injected with glucose-H3.

It is known that colonic goblet cells utilize glucose to synthesize the carbohydrate portion of mucus glycoprotein. To determine the intracellular site of this synthesis, glucose-H(3) was injected into 10-g rats. At 5, 20, 40 min, 1, 1(1/2), and 4 hr after injection, segments of colon were fixed and prepared for electron microscope radioautography. By 5 min after injection, label had been incorporated into substances present in the flattened saccules of the Golgi complex. At 20 min, both Golgi saccules and nearby mucigen granules were labeled. By 40 min, mucigen granules carried almost all detectable radioactivity. Between 1 and 4 hr, these labeled granules migrated from the supranuclear region to the apical membrane; here, they were extruded singly, retaining their limiting membrane. The evidence indicates that the Golgi saccule is the site where complex carbohydrate is synthesized and is added to immigrant protein to form the complete glycoprotein of mucus. The Golgi saccule, distended by this material, becomes mucigen granules. It is roughly estimated that one saccule is released by each Golgi stack every 2 to 4 min: a conclusion implying continuous renewal of Golgi stacks. It appears that the Golgi synthesis, intracellular migration, and release of mucus glycoprotein occur continually throughout the life of the goblet cell.

Amylases↗
PubMed1967

Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle.

Tritiated diisopropylfluorophosphate (DFP) was used to phosphorylate acetylcholinesterase (AChase) in the motor end plate of mouse sternomastoid muscle, and its distribution within the end plate was evaluated quantitatively by electron microscope radioautography. With the use of emulsion layers whose sensitivity to tritium had been calibrated, the density of AChase in different components of the end plate was calculated. The AChase was primarily localized (85%) in the junctional fold region. The concentration of AChase there was more than 20,000 active sites per cubic micron of tissue. The resolution of the technique was not sufficient to determine whether there was some AChase in the nerve end bulb; however, if there is any there, the concentration must be less than 10% of that at the junctional fold region.

Acetylcholinesterase↗