EM Autoradiography Research Articles

Impaired axonal regeneration in acrylamide intoxication.

AbstractAcrylamide is a neurotoxin known to impair regeneration of axons following nerve crush and to produce structurally abnormal regenerating sprouts. To investigate the mechanism of these abnormalities, protein synthesis and fast axonal transport were studied in acrylamide‐intoxicated and control rats 2 weeks after sciatic nerve crush. Using an in vitro preparation of sciatic nerve‐dorsal root ganglion, there was no difference in ganglion 3H‐leucine incorporation between the two groups. In these preparations of sensory axons, as well as in motor axons studied in vivo, a smaller proportion of rapidly transported radioactivity was carried beyond the crush in the acrylamide‐regenerating nerves compared to the control‐regenerating nerves. Correlative ultrastructural studies demonstrated that this difference reflected the impaired outgrowth of the acrylamide‐regenerating nerves, rather than an abnormality in fast transport. The acrylamide‐treated sprouts often developed swellings filled with whorls of neurofilaments; in addition, many sprouts ended in massively enlarged growth cones containing membranous organelles. EM autoradiography showed labeled, rapidly transported organelles accumulated in the neurofilamentous whorls, and therefore suggested that these organelles might be “trapped” or impeded in passage through these regions. However, there was no evidence that the growth cones received insufficient amounts of transported protein; in fact, the distended endings were densely labeled and apparently “ballooned” by transported organelles.These results suggest that acrylamide intoxication does not impair regeneration by diminishing the delivery of rapidly transported materials to the growing tip. Rather, the marked distention of the growth cones is interpreted as the morphological consequence of continued delivery of rapidly transported organelles into sprouts unable to utilize them in outgrowth.

Axonal transport of labeled material into sensory nerve endings of cat carotid body

The origin of nerve endings on glomus cells in the carotid body has been the subject of much controversy in recent years. Specifically, the problem is whether these nerve endings, which contain clear-core (“synaptic”) vesicles and mitochondria, arise from sensory neurons in the petrosal ganglion or from efferent neurons located in the brain stem or elsewhere. To study this problem, [ 3H]proline was applied to cat petrosal ganglia, the animals were allowed to survive for 3 h-7 days, and the peripheral distribution of the label was analyzed by sample oxidation/scintillation counting and by EM autoradiography. The time courses of distribution of label along the nerves and the accumulation of label in the carotid body indicated the presence of fast, intermediate and slow components of axonal flow. EM autoradiographs of carotid bodies showed the label localized almost exclusively to nerve fibers and to nerve terminals on glomus cells. As much as 60–90% of the nerve terminals were labeled in a given ultrathin-section autoradiograph. Passive movement of label from the injection site, or fibers of passage (efferent) through the ganglion, did not contribute to the labeled material since administration of [ 3H]proline onto the desheathed nerve away from the ganglion was ineffective in labeling the carotid body. The results suggest that most, if not all, nerve terminals on glomus cells in cat carotid body arise from neurons in the petrosal ganglion.

DNA-synthesizing cells in human fetal thymus

Fragments and suspensions of human fetal thymus were incubated in the presence of 3H-TdR to permit study of the distribution and morphology of DNA-synthesizing cells. Results of light and EM autoradiography showed that 1. although DNA-synthesizing cells were present in the medulla, the vast majority of these cells were localized in the thymic cortex, 2. cells with the typical EM appearance of small lymphocytes and lymphoid blast cells both synthesized DNA, and 3. cells in S-phase were predominantly 8 to 12 mum in size.

Intracellular distribution of lipophilic fluorescent probes in mammalian cells

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and 2-p- toluidinyl-6-naphthalene sulfonate (TNS)) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.

Morphology of rat's AV thalamic nucleus in light and electron microscopy

The structure of the rat's antero-ventral thalamic (AV Th) nucleus has been investigated in order to provide background information for the accompanying study in which an attempt was made to identify the synaptic terminals of the different afferent fiber systems to this nucleus by means of both EM autoradiography and the EM degeneration techniques. Nissl stained sections showed that the rat's AV Th nucleus contains mainly relatively light staining neurons which in Golgi material were found to possess tufted dendrites. In EM material three types of synaptic terminals were found which showed a topical distribution over the neuronal surface. Soma and stem dendrites carry a limited number of terminals with symmetrical synapses and flattened vesicles. Proximal dendrites carry mainly large asymmetrical synaptic terminals while distal dendrites are crowded with small asymmetrical synaptic terminals.

Electron microscopic autoradiography of isolated nucleolar transcription units

The transcription process is conveniently studied in oocyte nucleolar transcription units by means of quantitative EM autoradiography. There is a functional variability in the distribution of activity among the units. The distribution of activity within the units can also be determined by quantitative analysis, and was shown to depend on the length of the RNA fibrils.

EM autoradiography of non-sectioned biological material.

Methods of EM autoradiography suitable for the study of non-sectioned biological material deposited on a support film from suspension are described. The methods, which include modified procedures for specimen support and emulsion preparation, may also have advantages for the study of any specimen where physical separation of specimen and emulsion is desirable. In a study of the growth of 125I-labelled collagen fibrils reconstituted from solution in vitro, these procedures have allowed the production of EM autoradiographs which show a high resolution and are free from stress artefacts and chemography.

Problems of Understanding the Substructure of Synapses

This chapter discusses the morphological aspects of synapses that are thought to have a chemical mode of transmission. Using special prefixation and fixation techniques on the synapses of the rat central nervous system (cerebral and cerebellar cortices) and posterior pituitary endings, and frog forebrain, retina and motor endplate, microtubules can be observed focused on the terminal membrane. Synaptic vesicles can usually be observed associated with the microtubules except in the rat pituitary and frog retina. The present results give strong support to Schmitt's original hypothesis that synaptic vesicles slide along the microtubules and so arrive at the active zone of the presynaptic membrane. Therefore, now there is a morphological explanation of the clustering of synaptic vesicles at the appropriate zone of the presynaptic membrane for transmitter release. Monoaminergic synapses are, without doubt, the best characterized synapses at currently; the detection of the amine by the means of fluorescence microscopy, the presence of a dense core in a percentage of relatively small synaptic vesicles, the depletion or prevention of the uptake of the amine by a variety of drugs, the uptake of false transmitters—5- and 6-hydroxydopamine (the latter causing specific degeneration, and the uptake of tritiated noradrenaline, which can be detected by EM autoradiography, all provide important clues for identification.

In vivo recovery of muscle contraction after alpha-bungarotoxin binding.

Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.

3H leucine incorporation into myofibrils of normal and dystrophic mouse skeletal muscle.

The study of 3H leucine incorporation into skeletal muscle of mouse muscular dystrophy (129 ReJ/dy Bar Harbour strain) shows the uptake of isotope into myofibrils. The techniques employed were light and EM autoradiography before and after glycerination (Szent-Gyorgyi 1947). The results indicate a marked drop in uptake of the 3H-Leucine into myofibrils in the dystrophic animals, supporting the contention of Nihei et al (1971) that reduced myosin synthesis occurs in mouse muscular dystrophy.

Uptake of 35S-sulfate by morphologically differentiated replicating chondrocytes in Vivo: A double isotope electron microscope autoradiographic study

Developing cartilage in the 27-day regenerating forelimb of the newt Triturus viridescens was labeled with both 3H-thymidine and 35S-sulfate. The nuclear and cytoplasmic label was assessed quantitatively by EM autoradiography. It was found that chondrocytes incorporated 3H-thymidine into their nuclei while simultaneously incorporating 35S-sulfate in their cytoplasm. The incorporation of 35S-sulfate occurred primarily in the Golgi complex of morphologically differentiated chondrocytes at the base of the developing cartilage, and the 35S uptake by the DNA replicating chondrocytes was quantitatively equivalent per unit area of Golgi to that by the nonreplicating cells.

EM autoradiographic studies on polytene nuclei of Drosophila melanogaster: II. Organization and transcriptive activity of the chromocentre

The organization and the transcriptive activity of the chromocentre region of salivary gland nuclei of Drosophila melanogaster have been examined by electron microscopy and EM autoradiography. The heterochromatic chromocentre region is organized into a central compact block of chromatin, surrounded by a large number of smaller, interconnected blocks. The latter are continued into the euchromatic chromosome arms. The two types of chromatin organization in the chromocentre are reminiscent of the classical α- and β-heterochromatin. Two types of ribonucleoprotein-like particles regularly occur in the β-heterochromatin. EM autoradiography after short (5 min) in vitro pulse of 3H-uridine shows that the β-heterochromatin is as active in RNA synthesis as the euchromatin in the nucleus; the α-heterochromatin is completely inactive. It is suggested that the two types of particles seen in the β-heterochromatin are products of RNA synthesis occurring in this region. These observations raise doubts on the general validity of the notion of total inactivity of constitutive heterochromatin.

Studies on the ship-fouling algaEnteromorpha

A cytochemical technique for detection of polysaccharides indicates that the Golgi-derived adhesive vesicles in swimming zoospores contain glycoprotein. The carbohydrate moiety appears to be polymerized after the vesicles have become detached from the Golgi apparatus. EM autoradiography usingL-leucine-4,5-3H shows that protein synthesized in the ER passes through the Golgi complex into the adhesive vesicles. On settlement the vesicles discharge their content as a fibrillar attaching material.

EM autoradiographic studies on polytene nuclei of Drosophila melanogaster. 3. Localisation of non-replicating chromatin in the chromocentre heterochromatin.

Replication in the chromocentre heterochromatin of salivary gland polytene nuclei of Drosophila melanogaster has been examined by 3H-thymidine EM autoradiography. In vitro pulse labelling of salivary glands from late third instar larvae showed that the chromocentre heterochromatin replicates in synchrony with the euchromatin in the nucleus. Within the chromocentre region, the central compact mass, identified earlier as the alpha heterochromatin, did not incorporate 3H-thymidine at any stage of the S, while the surrounding beta heterochromatin was always labelled in nuclei with labelled euchromatin. In a second set of experiments, growing larvae from just after hatching till late third instar stages, were fed on food containing 3H-thymidine, and at the end of larval life, the incorporation in salivary gland nuclei was examined by EM autoradiography. A grain density analysis of the EM autoradiographs revealed that the alpha heterochromatin does not replicate at all from after hatching till late third instar while the beta heterochromatin replicates as much as the euchromatin. Non-replication of the alpha heterochromatin provides the explanation for the lowered amount of heterochromatin in the polytene nuclei compared to their diploid counterparts. Implications of these observations on the organization of chromocentre heterochromatin in polytene nuclei and its homology to the heterochromatic regions in mitotic chromosomes are discussed.

Reconsideration of resolution in EM autoradiography using a biological line source

SUMMARYPieces of integument containing cuticle and hypodermal cells from the desert locust Schistocerca gregaria were pulse‐labelled to produce very narrow bands of radioactive chitin or resilin in the cuticle. Araldite sections of such cuticle were used as a radioactive line source to test the resolution and the possible effect of self‐absorption due to heavy metal staining of tissue‐sections in EM autoradiographs.The approximate position of the source and the half‐distance of resolution (HD) in EM autoradiographs were determined by replotting the grain‐distribution in integrated form, producing a sigmoid‐shaped curve. The values of HD, the position of the source and the total number of grains were then used to superimpose a theoretical grain‐density distribution function on the experimental grain‐distribution histograms. The results confirm the line or very narrow band nature of the radioactive chitin or resilin in the tissue‐sections used.When the source was resilin situated in the cuticle at about 1 μm from the cell‐surface, the values for HD were 130–180 nm in Ilford L4 and about 100 nm in Agfa‐Gevaert NUC 307. When the source was chitin situated just outside the cell surface, the corresponding value for HD in Ilford L4 was 85–125 nm. Possible adverse effects of electron beam damage on resolution in resilin‐containing samples are discussed.In sections with radioactive chitin at the cell surface, the total number of grains in the cell‐side of the histogram was always significantly lower than that in the cuticle‐side. This marked asymmetry in grain distribution is considered to be indicative of much greater self absorption of beta‐particles from the tritium source, in heavily stained areas of the sections.

Distribution of DNA replicator sites in mammalian nuclei: II. Effects of prolonged inhibition of DNA synthesis

The effect of prolonged treatment of CHO Chinese hamster or Microtus agrestis cells with amethopterin or 5-fluorodeoxyuridine on the DNA replicator site distribution and on cell survival has been investigated with EM autoradiography of nuclear sections and cell cycle studies. A pool of DNA precursors in the CHO cells allows slow progression of G1 cells through the first S period with a normal homogeneous replicator site distribution in early-S, cell survival is normal. After mitosis cells are blocked at the beginning of the second S period with replicator sites now distributed on the nuclear membrane and nucleolar periphery. Degenerative changes rapidly occur, cell recovery is delayed and survival is reduced on release from the inhibition. Microtus cells with no pool of precursors show normal replicator site distribution when labelled in exponential growth. After inhibition however, cells do not progress through the S period and show early peripheral distribution of replicator sites and early degenerative changes. This peripheral distribution probably represents DNA breakdown. It is proposed that small replicating fragments are produced, and accumulate on the nuclear membrane and around the nucleolus during the process of thymineless death.

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy

SUMMARYThe loss of 14C ethanolamine‐ and 3H choline‐labelled phospholipids from rat liver during tissue preparation for electron microscopy has been examined. Column and thin‐layer chromatography combined with double‐label scintillation spectrometry were used to analyse the radioactive phospholipid content of the livers of rats injected simultaneously with 14C aminoethanol and 3H choline chloride. After 4 h (in vivo) the 14C and 3H labels were mainly incorporated into phosphatidyl ethanolamine and phosphatidyl choline respectively but some 14C label had been incorporated into phosphatidyl choline. Chopped rat liver was fixed in glutaraldehyde or osmium tetroxide or both sequentially and tissues were dehydrated in ethanol and embedded in Araldite. In each procedure examined the choline label proved more labile than the ethanolamine. After glutaraldehyde fixation alone complete loss of phosphatidyl choline occurred and half of the phosphatidyl ethanolamine was also lost. Following osmium tetroxide fixation negligible loss of either phosphatide occurred. In terms of phospholipid retention, no advantage was gained by glutaraldehyde fixation prior to osmium tetroxide fixation. The results show that both ethanols and embedding monomers are potent phospholipid solvents. The data also suggests that EM autoradiography of these two phosphatides may be carried out with reasonable confidence although it must be pointed out that a high degree of retention does not necessarily imply retention in situ.