Radioautographic Reaction Research Articles

In male mouse germ cells, copper-zinc superoxide dismutase utilizes alternative promoters that produce multiple transcripts with different translation potential.

Copper-zinc superoxide dismutase (SOD-1) is an enzyme that is widely expressed in eukaryotic cells and performs a vital role in protecting cells against free radical damage. In mouse testis, three different sizes of SOD-1 mRNAs of about 0.73, 0.80, and 0.93 kilobases (kb) are detected. The 0.73-kb mRNA is found in early stages of male germ cells and in all somatic tissues. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase H digestions and Northern blot analyses reveal that the three SOD-1 mRNAs are derived from two transcripts, a ubiquitously expressed transcript and a post-meiotic transcript, which differ by 114-120 nucleotides. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5'-untranslated region. Using a probe derived from the 5'-untranslated region of the 0.93-kb SOD-1 mRNA, we have established that it originates from an alternative upstream promoter contiguous with the somatic SOD-1 promoter. Polysomal gradient analysis of the three mouse testis SOD-1 mRNAs reveals that the 0.93-kb SOD-1 mRNA is primarily non-polysomal, while the 0.80- and 0.73-kb SOD-1 mRNAs are mostly polysome associated. A faster migrating form of the 0.93-kb SOD-1 mRNA is present on polysomes as a result of partial deadenylation. In a cell-free translation system, the 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-1 mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-1 mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-1 mRNAs undergo adenylation changes, and one of the post-meiotic SOD-1 mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form.

Enkephalin-containing nerve cell bodies in the substantia nigra of the rat: Demonstration by immunohistochemistry and in situ hybridization

The use of a new and very sensitive immunohistochemical method, combined with intracerebral injections of colchicine, has allowed us to show that a number of nerve cell bodies immunoreactive for Met-enkephalin are present in several mesencephalic nuclei of the rat, including the different subdivisions of the substantia nigra (SN). The existence of numerous neuronal somata of this kind in the medial part of the SN pars compacta and in the lateral half of the pars reticulata is rather new. The latter has been ascertained by demonstrating a perikaryal immunoreactivity for synenkephalin in the same regions of the SN. In addition, by in situ hybridization, we have shown that neuronal cell bodies expressing the preproenkephalin A (PPA) gene are also present in the same regions of the SN. However, the fact that a strong radioautographic reaction was found only in rats which received an intranigral injection of 6-hydroxydopamine indicates that these neurons are probably not dopaminergic and that an induction of the PPA gene occurs in these animals.

Prostaglandin E2 Receptors in the Chicken Spinal Cord: 2. Radioautographic Localization.

High and low affinity binding sites for PGE2 were localized at the cellular level by means of radioautography. Two types of radioautographic reactions were observed in cryostat sections incubated with 0.6 nM [3H]PGE2: (i) a diffuse reaction scattered over the neuropil; (ii) a stronger reaction over the perikarya of some motoneurons. However, not all the motoneurons were equally labelled; the density of silver grains increased 12 times from poorly labelled to highly labelled cells. After incubation with an excess of unlabelled PGE2 the labelling was drastically reduced over motoneurons and the neuropil, so that the specific binding corresponded to 90 and 85% respectively of the total binding. Two facts indicated that the motoneurons possessed mainly high affinity binding sites which could be saturated by PGE2 concentrations in the nanomolar range: (i) a five-fold increase of [3H]PGE2 concentration (from 0.6 to 3 nM) produced only a 1.5 times increase in the density of silver grains over the perikarya; (ii) incubation in presence of 14 nM unlabelled PGE2 induced a drastic reduction of 0.6 nM [3H]PGE2 binding to the motoneurons. The presence of high affinity binding sites in motoneurons suggests that PGE2 could act as a modulator of spinal reflexes.

Nucleolar structure and synthetic activity during meiotic prophase and spermiogenesis in the rat

The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli as examined in rats sacrificed 1 hr after intratesticular injection of 3H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords--a nucleolonema--within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extranucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 3H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the second maturation division do not synthesize rRNA.

Retrograde axonal transport specificity in the locus coeruleus neurons after [ 3H]noradrenaline injection into the rat olfactory bulb

Retrograde axonal transport process was investigated in the afferent systems to the rat olfactory bulb, after [ 3H]noradrenaline ([ 3H]NA) injection into the olfactory bulb, in order to provide evidence regarding its specificity and the biochemical basis supporting this specificity. Radioautographs showed that [ 3H]NA unilaterally injected into the olfactory bulb at a concentration of 10 −3 M, resulted in labeling of the structures afferent to the olfactory bulb, mainly on the injected side: locus coeruleus (LC), dorsal and central raphes, nucleus of the lateral olfactory tract and piriform cortex. Heavy labeling was observed on the noradrenergic LC cell bodies, whereas the radioautographic reaction was less intense on the other structures. After 10 −4 M injection, the labeling intensity of the LC cell bodies was lower while very rare weakly labeled cell bodies persisted in the dorsal raphe, nucleus of the lateral olfactory tract and piriform cortex. The LC cell bodies were exclusively labeled when the concentration of [ 3H]NA injection was 10 −5 M. All the other structures were devoid of labeling. It was still possible to detect labeled cell bodies in the LC for a 10 −6 M concentration. Following bilateral injections of [ 3H]NA (10 −3 M) the total radioactivity retrogradely transported to the LC represented about 4 times the total radioactivity measured in the periaqueductal gray substance (as control tissue of the tracer diffusion). Fractional study by ethanol of LC tissue homogenate and liquid scintillation counting of each fraction showed that 60% of the total radioactivity (about 2.5 times the control value) was in the supernatant and 40% (about 20 times the control value) was associated with the precipitate. In the other regions such as the dorsal and central raphes and periaqueductal gray substance, radioactivity was mainly found in the supernatant, except for the dorsal raphe whose precipitate contained a low amount of radioactivity (about 4 times the control value). Colchicine (an axonal transport inhibitor) bilaterally injected into the medial forebrain bundle and systemic administration of desipramine (a noradrenaline uptake inhibitor) decreased the radioactivity associated with the LC precipitate by 90 and 85% and the LC supernatant radioactivity by 55 and 35%, respectively. These pretreatments did not significantly affect the radioactivity amounts measured in the different fractions of dorsal and central raphes and periaqueductal gray substance. Radioautographic study after colchicine treatment showed a large decrease in the labeling intensity of the LC cell bodies as compared to the non-treated side. Therefore, we suggest that low concentrations (10 −5 M) of [ 3H]NA injected in the olfactory bulb determine specific conditions of noradrenergic pathway labeling. This specific labeling after migration could be supported by the radioactive ethanol precipitate which would appear to contain [ 3H]NA- and/or 3H-derivatives-binding protein. Such a 3H-macromolecular complex, which could represent the specific carrier, may well undergo retrograde transport from the nerve terminals towards the cell bodies.

Binding and internalization in vivo of [125I]hCG in Leydig cells of the rat.

The present study was performed to demonstrate the binding, mode of uptake, pathway and fate of iodinated human chorionic gonadotropin ([125I]hCG) by Leydig cells in vivo using electron microscope radioautography. Following a single injection of [125I]hCG into the interstitial space of the testis, the animals were fixed by perfusion with glutaraldehyde at 20 minutes, 1, 3, 6 and 24 hours. The electron microscope radioautographs demonstrated a prominent and qualitatively similar binding of the labeled hCG on the microvillar processes of the Leydig cells at 20 minutes, 1, 3, and 6 hours. The specificity of the [125I]hCG binding was determined by injecting a 100-fold excess of unlabeled hormone concurrently with the labeled hormone. Under these conditions, the surface, including the microvillar processes of Leydig cells, was virtually unlabeled, indicating that the binding was specific and receptor-mediated. In animals injected with labeled hCG and sacrificed 20 minutes later, silver grains were also seen overlying the limiting membrane of large, uncoated surface invaginations and large subsurface vacuoles with an electron-lucent content referred to as endosomes. A radioautographic reaction was also seen within multivesicular bodies with a pale stained matrix. At 1 hour, silver grains appeared over dense multivesicular bodies and occasionally over secondary lysosomes, in addition to the structures mentioned above, while at 3 and 6 hours, an increasing number of secondary lysosomes became labeled. At 24 hours, binding of [125I]hCG to the microvillar processes of Leydig cells persisted but was diminished, although a few endosomes, multivesicular bodies and secondary lysosomes still showed a radioautographic reaction. No membranous tubules that were seen in close proximity to, or in continuity with, endosomes and multivesicular bodies were observed to be labeled at any time interval. Likewise, an attempt to correlate silver grains with small coated or uncoated pits, the stacks of saccules of the Golgi apparatus and other Golgi-related elements including GERL, proved unsuccessful, since these structures were mostly unlabeled. These in vivo experiments thus demonstrate the specific binding of [125I]hCG to the plasma membrane of Leydig cells predominantly on their microvillar processes, and the subsequent internalization of the labeled hCG to secondary lysosomes. In addition, binding and internalization of hCG persisted for long periods of time.

Use of slide-mounted frozen sections to study the autoradiographic localization, characterization and quantification of luteinizing hormone-releasing hormone receptors in the rat pituitary

Slide-mounted frozen sections were used for the autoradiographic localization, characterization and quantification of LHRH receptors. The radioligand was the iodinated LHRH agonist [D-Ser(TBU) 6, des-Gly-NH 2 10]LHRH ethylamide (LHRH-A). Specificity studies demonstrated that specific labeling was concentrated in the anterior pituitary lobe and that the tracer could be displaced with either LHRH or LHRH-A. Scatchard analysis of saturation curves gave one-site interactions with a high affinity (Kd = 0.48 ± 0.1 nM) for the iodinated LHRH-A. In order to evaluate the effects of different treatments on the LHRH receptor concentration, the effects of sex steroids on the density of radioautographic reaction were evaluated using an image analyzer. The results obtained were similar to those previously reported with classical binding assays. Thus, this simple technique appears suitable for the study of localization and characterization of LHRH receptors in different experimental conditions.

Transport of iron and transferrin synthesis by the seminiferous epithelium of the rat in vivo.

The transport of radioactive iron across the seminiferous tubules was analyzed in vivo by light-microscope quantitative radioautography. At 5 min after a single intratesticular injection of 55Fe-transferrin, a strong labeling of the basal aspect of the seminiferous epithelium was observed. Between 30 min and 2 h, the labeling on the basal aspect of the seminiferous epithelium decreased. This decrease was accompanied by a substantial increase of the radioautographic reaction over the cellular elements in the adluminal compartment. These results were consistent with the demonstration of 59Fe associated with meiotic spermatocytes and differentiating spermatids isolated by velocity sedimentation from testes injected with 59Fe-transferrin. Furthermore, after a single intratesticular injection of 59Fe-labeled human transferrin, radiolabeled rat transferrin was immunoprecipitated from homogenates of isolated tubules with a specific antibody and appeared as a single radioactive band on fluorographs of urea/polyacrylamide gels. Similarly, 59Fe-labeled rat transferrin but not 125I-transferrin was immunoprecipitated from rete testis fluids of testes infused with either 59Fe- or 125I-labeled human transferrin. Finally, the synthesis of testicular transferrin in vivo was demonstrated in fluorographs of immunoprecipitated transferrin after an intratesticular injection of 35S-methionine in rats whose livers were excluded from the general circulation by ligation of both the hepatic artery and the portal vein. Thus, our results demonstrated a unidirectional system of iron transport from the basal compartment of the seminiferous epithelium to the germ cells in the adluminal compartment involving two distinct transferrins, i.e., a serum transferrin and a testicular transferrin synthesized by the seminiferous epithelium.

Immunohistochemical and radioautographic evidence of monoamine-containing cells in bioluminescent elytra of the scale-worm Harmothoe imbricata (Polychaeta)

Elytra of the scale-worm Harmothoe imbricata were examined for the presence of monoamine-like immunoreactivities and radioautographic reactions. Serotonin (5-HT)-like immunoreactivity was widely distributed among the cellular constituents of the elytra, being present in epithelial cells including photocytes, in elytral nerves, clear cells and the loose neuronal plexus of the middle compartment. The distribution of [3H]5-HT labelling coincided with that of the immunoreactivity except for an additional reactive band extending through the upper cuticle layer. Tyrosine hydroxylase (TH)-like immunoreactivity was detected in epithelial cells, sensory papillae and elytral ganglion and nerves, with little or no staining in clear cells and plexus neurons of the middle compartment. Radioautographic labelling with [3H]noradrenaline and [3H]adrenaline overlaid many epithelial cells, elytral nerves and sensory papillae, but not the loose neuronal plexus or, apparently, clear cells. It is concluded that monoaminergic systems are widely distributed and that they must play important roles as neuroactive and/or paracrine substances in the elytral neuroectoderm. The distribution of [3H]5-HT label in photocytes also suggests the involvement of serotonergic mechanisms in luminescence control, luminescence being the only known effector activity of elytra.

Receptor-mediated endocytosis of transferrin by Sertoli cells of the rat.

Binding of 125I-transferrin (125I-Tf) to the plasma membrane of Sertoli cells and its endocytosis were analyzed by means of light- and electron-microscope quantitative radioautography. Five minutes after 125I-Tf was injected into the interstitial space of the testis, a strong labeling of the basal aspect of the seminiferous epithelium was observed in light-microscope radioautographs. Injection of the same dose of 125I-Tf plus a 200-fold excess of cold transferrin resulted in a marked diminution of the radioautographic reaction, indicating that the initial strong labeling with radiolabeled transferrin was specific. These results were consistent with the localization of immunoreactive fluorescence of transferrin receptor at the base of the seminiferous epithelium. In electron-microscope radioautographs of tubules collected at 5 min after injection, the membrane of Sertoli cells facing the basement membrane was well labeled with 125I-Tf. At 15 and 30 min, the plasma membrane was less intensely labeled, but the silver grains were then seen overlying multivesicular bodies with an electron-lucent matrix, identified as endosomes. This population of endosomes was always seen at a short distance from the basal membrane of Sertoli cells. At 90 min, no more labeling of the plasma membrane, endosomes, or any other cytoplasmic component was observed. Isolated seminiferous tubules and Sertoli cells labeled with 125I-Tf at 4 degrees C were rinsed and reincubated in a label-free medium at 37 degrees C for various periods of time from 5 to 90 min. A radioactive protein precipitated by trichloroacetic acid, presumably intact transferrin, was released from the tubules into the incubating medium; when measured, it was found to increase rapidly from 5 to 45 min and stabilize thereafter. These results suggest that transferrin was internalized by receptor-mediated endocytosis, reached endosomes, and then was released to the extratubular space. When native ferritin (NF), a tracer for fluid-phase endocytosis, was infused within the lumen of seminiferous tubules and 125I-Tf was simultaneously injected into the interstitial space, both markers rapidly reached different populations of endosomes. Endosomes labeled with NF, scattered throughout the cytoplasm, evolved with time into dense multivesicular bodies and secondary lysosomes, whereas radiolabeled transferrin reached only the endosomes located in the basal cytoplasm of Sertoli cells. The latter thus appeared to be principally involved in the uptake and recycling of transferrin.

Distribution of epidermal growth factor receptors in the rat ovary

The distribution of epidermal growth tactor (EGF) was studied in the ovary using light microscope radioautography which was performed at different time intervals (2–60 min) after intravenous (i.v.) injection of [ 125I]EGF into adult rat at random stages of the estrous cycle and also after topical localization of iodinated EGF on slide-mounted frozen ovarian sections. After i.v. injection of label, the labeling was mostly observed in the theca interna cells of secondary, preovulatory and atretic follicles and luteal cells of corpus luteum. Primordial and primary follicles did not show any significant labeling. The time-course study performed on luteal cells showed that, 2 min after injection, most silver grains were found at the periphery of the cells. At the 10 min time interval, silver grains were found both at the periphery and over the cytoplasm of these cells. The number of grains was very reduced over the cytoplasm at the 60 min time interval. In the in vitro study, a positive radioautographic reaction was seen in the same cellular elements as found in vivo, with the additional labeling of the granulosa cells of growing and preovulatory follicles. Control experiments indicated that the radioautographic labeling was due to specific interaction of [ 125I]EGF with its receptor. These results clearly indicate that EGF binding sites are present in luteal, thecal and granulosa cells, and provide support for the inhibitory and stimulatory actions of EGF on different parameters of ovarian cells.

Radioautographic localization of 125I-atrial natriuretic factor binding sites in the brain.

Rats were injected through the carotid artery (cephalad direction) with 18.9 mu Ci of either 125I-Arg 101-Tyr 126 atrial natriuretic factor alone or together with an excess of unlabeled hormone. At 2 min after injection, all rats were fixed in vivo by perfusion and serial sections of the whole brain were processed for light microscope radioautography. The radioautographic reaction produced by 125I-atrial natriuretic factor alone was localized in all circumventricular organs (except the subcommissural organ), the smooth muscle cells and endothelial cells of arteries, arterioles, veins, venules, the endothelial cells of intraparenchymal capillaries and the epithelial cells of the choroid plexus. In rats which received 125I-atrial natriuretic factor plus an excess of unlabeled hormone, the radioautographic reaction was reduced by 70-90%. Binding sites are thus localized in regions of the brain, some of them involved in the central monitoring of blood pressure and osmolarity. In addition, the presence of binding sites in the cerebral vasculature and in the epithelium of the choroid plexus suggests that circulating ANF may play a role in the control of cerebral blood flow and in the production of vertebrospinal fluid.

In vitro uptake and metabolism of [3H]indole compounds in the pineal organ of the pike. II. A radioautographic study.

In a previous radiobiochemical study [Falcón et al., 1985] it was demonstrated that the pineal organ of the pike incubated in vitro takes up and metabolizes [3H]serotonin and [3H]melatonin. Parallel to this experiment (present work), pineal organs were investigated by use of light and electron microscopic radioautography to investigate the cell types involved in indole metabolism. Irrespective of the tracer and experimental conditions used, the radioautographic reactions were mainly found in cone-like and modified photoreceptor cells of the pineal parenchyma. Taking into account the limits of the method used as well as previous data concerning the direct photosensitivity of the organ, the localization of exogenous indoles, and the localization and circadian variations of endogenous ones, it is concluded that the photoperiodic information is translated into both nervous and indolic signals in cone-like photoreceptor cells and into indolic signals in modified photoreceptor cells.

Biochemical and electron microscope radioautographic study of intestinal absorption of tritiated palmitic and oleic acids in control and actidione-cycloheximide-treated rats.

Intestinal absorption of tritiated palmitic and oleic acids was investigated in control and actidione-cycloheximide-treated rats. Pancreatic juice was collected and 24 hr later an intestinal loop was cannulated in situ. A 90 mumole-lipid emulsion composed of an equimolar mixture of monopalmitin, palmitic and oleic acids with bile was infused with either 3H-labeled palmitic or oleic acid. After 15 or 30 min biochemical analysis was carried out to follow the uptake and the transfer of the labeled fatty acids. Mucosa was removed for both biochemical analysis of lipid classes and the ultrastructural or radioautographic electron microscope study depending on the radioactive activity. Following actidione-cycloheximide treatment, the uptake of both fatty acids decreased, but the amount of lipids in the mucosa greatly increased, while amount of the reesterification of fatty acids in the mucosa diminished. Consequently the amount of infused palmitic acid which was transferred in 15 min decreased from 36% in the absence of treatment to 4.6% when treatment was used. The corresponding figures for oleic acid are 70% and 20.9%. Unstructured lipids were in the intercellular spaces, indicating that a cytotoxic effect had occurred which produced a defect in the lipoprotein particle organization. The Golgi complex, in the final step of the chylomicron synthesis before exocytosis, showed low level of radioautographic reaction indicating less participation by the complex in lipid transfer. The various processes which were inhibited during long-chain fatty acid absorption from the luminal area to the Golgi complex included fatty acid binding by proteins, enzymatic acylation and esterification, apoprotein participation. This inhibition explains why long-chain fatty acid absorption was greatly impaired. Moreover, our observations compared with those obtained during decanoic acid absorption, particularly our radioautography based observations, emphasize the important role of the Golgi complex which requires intense membrane turnover during lipid absorption.

An improved method for freeze-fracture radioautography of tissues and cells, as applied to duodenal epithelium and thymic lymphocytes.

An improved method has been devised for the localization of radioactive substances to either one of the leaflets of cellular membranes. After tissue specimens are freeze-fractured and covered with a platinum-carbon replica, they are freeze-dried to allow coating with radioautographic emulsion at room temperature. After exposure at 4 degrees C and development, the emulsion is protected by layers of carbon and grease before the tissue underlying the replica is dissolved in sodium hypochlorite. The grease is removed in Freon 14 and the replica with its emulsion cover is mounted on a specimen grid for electron microscopic examination. The accuracy of radioactivity localization was demonstrated using 3H-thymidine-labeled liver by finding silver grains over the same sites after freeze-fracture as after thin section radioautography. Tests with 3H-methacrylate revealed that the interposition of a platinum-carbon replica decreased the radioautographic reaction by over 80%; hence, the need for long exposure. Only 67% of the silver grains came from radiation sources located beyond the upper 0.05 micron of the specimen and, therefore, the emulsion could be affected by radiation sources located not only within membrane leaflets but also in nearby cytoplasm. Thus, when 3H-fucose was injected into rats to locate newly formed glycoproteins within intestinal epithelium membranes, some of the silver grains found over E and P faces might be produced by radiation coming from the adjacent cytoplasm. To localize label within membrane leaflets in the absence of radiation sources in the cytoplasm, lymphocyte suspensions were incubated with 3H-concanavalin A at 0 degrees C. The plasmalemma radioactivity was then restricted to the two membrane leaflets, with 87-93% of the silver grains on the E leaflet and 7-13% on the P leaflet. It appears that, under these conditions, the technique provides adequate localization of radioactivity to the leaflets of the cell membrane.

Retention of indoles in the pineal organ of the bird (Melopsittacus undulatus, Shaw) during its preparation for radioautographic study

Since high-resolution radioautography (dipping technique) might be very useful for the study of indole metabolism in the pineal cells, the retention of [3H]-indoles has to be examined during the preparation of specimens for electron microscopy (EM). The pineal organ of the parakeet (Melopsittacus undulatus) was used in the present work. 1) Indole metabolism: following uptake of [3H]-5-hydroxytryptophan ([3H]-HW) in vivo and [3H]-5-hydroxytryptamine ([3H]-HT) in vitro in similar seasonal and nycthemeral conditions--all the known pineal indolic metabolites were recovered by thin layer chromatography. [3H]-5-methoxyindoles were also formed from [3H]-melatonin ([3H]-aMT). 2) The radioactivity of fluids used in the processing of pineal organs in EM was determined by liquid scintillation counting: (a) no exogenous [3H]-indoles could be revealed in EM solutions after [3H]-HW in vivo uptake. (b) 8.8 to 13.4% of [3H]-indoles were washed out by glutaraldehyde after [3H]-HT in vitro uptake. (c) most of the 5-methoxyindoles after in vitro uptake of [3H]-aMT were lost in glutaraldehyde. Our chromatography procedures did not permit the identification of [3H]-indoles extracted by the glutaraldehyde fixative. In previous experiments, [3H]-HW and [3H]-HT uptake showed the presence of selective radioautographic reactions in the cells of the receptor line; however, silver grains were scarce and diffusely distributed in the pineal parenchyma after [3H]-aMT uptake.

Axoplasmic incorporation of amino acids in a myelinated fiber exceeds that of its soma: A radioautographic study

The axoplasmic incorporation of amino acids was studied in the giant Mauthner axon to determine its magnitude, to estimate the half-life of the resulting material, and to establish whether or not it undergoes transport. The incorporation of labeled leucine, lysine, and proline was followed by radioautography. The tracers were injected away from the cell body, either in the floor of the IV ventricle or into the spinal cord. Radioautographic reaction on the Mauthner somata was barely discernible. However, at the site of injection, for survivals to 5.6 days, a peak of reaction was observed on the Mauthner axoplasm, with a sharp and symmetric decrement in both central and distal directions; the intensity of the peak decreased with survival (14% at 5.6 days). In intact fibers, the intensity of the reaction was 1.4% compared with that over the Nissl substance of neighboring somata; by contrast, in severed fibers whose axoplasm had been directly exposed to the tracers the reaction rose to 4.1%. At early times, the intensity of the response of the axoplasm as a function of survival of the fish did not have a lag time. In high-resolution radioautograms, most of the grains overlay the ground axoplasm. Cycloheximide depressed the reaction over the fiber open to the extracellular space by 55% whereas only by 20% over the intact fiber. Adsorption of free amino acids or their binding to tRNA were ruled out as a cause of the radioautographic response. Our results indicated that incorporation of amino acids into macromolecules of the Mauthner fiber occurred locally, and its magnitude over the whole axoplasm was estimated to be one order of magnitude larger than that occuring in the some. The bulk of the material resulting from this incorporation did not undergo transport and survived some days. We suggest that the amino acids were incorporated by a ribosomal mechanism.

Accumulation of [ 3H]fucose-labelled glycoproteins in the Golgi apparatus of dorsal root ganglion neurons during inhibition of fast axonal transport caused by exposure of the ganglion to Co 2+-containing or Ca 2+ -free medium

Previous in vitro studies have established that Co 2+-containing or Ca 2+-free media interfere with the initiation of the fast axonal transport of proteins. The present study has used light- and electron-microscope radioautography to compare the distribution of [ 3H]fucose-labelled glycoproteins in neuronal cell bodies of control dorsal root ganglia and ganglia incubated for 16–17 h in Ca 2+-free medium or in medium containing 0.18 mM Co 2+. The radioautographic reaction in control cell bodies was diffusely scattered throughout the cytoplasm; grain counts revealed that 22% of the reaction was associated with elements of the Golgi apparatus and 78% was over other organelles and the remainder of the cytoplasm. In most experimental-cell bodies, 78% of the silver grains were clustered over elements of the Golgi complex whereas other organelles and the remainder of the cytoplasm were comparatively much less labelled; structural alterations of the Golgi apparatus were also produced by the modified media. In parallel studies where the radioactivity in nerve trunks and ganglia was measured by liquid scintillation counting, it was found that the Ca 2+-free medium and the Co 2+-containing medium both reduced by approximately 80% the quantity of [ 3H]fucose-labelled glycoproteins which were carried by the fast axonal transport system; they did so without interfering with the incorporation of [ 3H]fucose into glycoproteins. The results indicate that in the presence of Co 2+ or in the absence of Ca 2+ the proteins which are destined for fast axonal transport accumulate at the Golgi apparatus of neuronal cell bodies. These results thus suggest that Ca 2+ is required for proteins to leave the Golgi region in transit to the fast axonal transport system.

High-resolution radioautographic study of dopamine binding sites in the rat neostriatum using 3H-domperidone.

The possibility to use the new ligand 3H-domperidone to identify some dopamine binding sites at the ultrastructural level was assessed in the neostriatum after in vivo administration and high-resolution radioautography. Since this ligand does not cross the blood-brain barrier, intracerebral injections were performed, which resulted in a gradient of diffusion of the tracer. According to increasing distances to the injection site, a quantitative study of the radioautographic reaction was realized. An intense and diffuse reaction took place in the vicinity of the injection site in control rats. On the contrary, numerous accumulations of silver grains were observed in the peripheral zone. The statistical analysis of the distribution of the clusters showed that they were more numerous over the contacts between nerve terminals and dendritic spines than expected from a distribution at random; moreover half of these labelled contacts were differentiated in synapses of the asymmetric type. When the animals were pretreated with haloperidol in order to block the dopaminergic binding sites, we found a decrease in the total number of the number of silver grains. A decrease in the number of clusters of silver grains was noted over nerve terminals and synaptic contacts in both peripheral zones while the nonspecific labelling was increased over other structures. We conclude to the possibility of the detection of the dopaminergic binding sites by electron microscopic radioautography. Moreover we confirm the existence of dopaminergic synapses in the neostriatum with this new technique.

Selective retrograde transport of 3H-serotonin in vagal afferents

A new serotonergic afferent vagal component has been demonstrated in the cat by radioautography. Twenty-four hours after a bilateral injection of tritiated serotonin ( 3H-5-HT) into the area of the nucleus of the solitary tract (NST), heavily and lightly labelled cell bodies were observed in the nodose ganglia. After unilateral injections of 3H-5-HT into the same area, labelled ganglionar cell bodies were found in the ipsilateral nodose ganglion. Some were also found in the contralateral one, suggesting a serotonergic crossed fibers component. Dense clusters of silver grains, depicting typical labelling of neuronal varicosities, were observed in the NST. After destruction of the serotonergic terminals with 5,7-dihydroxytryptamine, followed by injection of 3H-5-HT, the number of labelled cell bodies decreased dramatically in the ipsilateral nodose ganglia and the clusters of silver grains disappeared in the NST. After ligature or section of the supranodose vagal nerve, following injection of 3H-5-HT into the NST, no radioautographic reaction was observed in the homolateral nodose ganglia. The present study demonstrates the existence of a peripheral serotonergic system in vagal afferents. The physiological implications of this new serotonergic visceral pathway remain to be studied.