scholarly journals Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions

1976 ◽  
Vol 69 (1) ◽  
pp. 144-158 ◽  
Author(s):  
HC Fertuck ◽  
MM Salpeter
Keyword(s):  
Electron Microscope ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Molecular Organization ◽  
Reference Structure ◽  
Axonal Membrane ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Valid Criterion

The distribution and quantitation of (125)I-α-bungarotoxin (α- BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetantic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, α-BTX was bound to approximately 90 percent of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of α-BTX relative to this membrane was determined to be 46,000 +/- 27 percent sites/μm(2). By testing various hypothetical models the distribution of developed grains was found to be consistent with the hypothesis that the main acetylcholine-receptive surface coincides with the electron-dense, thickened portion of the junctional fold membrane situated at the juxtaneuronal region of the folds and dipping approximately 2,200 A be approximately 30,500 +/- 27 percent sites/μm(2) of this postsynaptic dense membrane. There was a sharp gradient in α-BTX binding extrajunctionally, with the concentration decreasing to approximately 4 percent of the subsynaptic value within 1μm from the edge of an axonal terminal (bouton) to <1 percent within 3μm and to <0.2 percent beyond 7 μm from that terminal. Below 4,000 A (i.e. half-way from the top of the junctional folds) the concentration of α-BTX was also about 3 percent of the peak subsynaptic value. The binding density at the bottom of the junctional folds is this comparable to extrajunctional sarcolemma at equal distance from a nerve terminal. The molecular organization at the neuromuscular junction relative to its function is discussed.

scholarly journals Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

1978 ◽  
Vol 78 (1) ◽  
pp. 274-285 ◽  
Author(s):  
M M Salpeter ◽  
A W Rogers ◽  
H Kasprzak ◽  
F A McHenry
Keyword(s):  
Electron Microscope ◽  
Surface Area ◽  
Binding Sites ◽  
Extraocular Muscle ◽  
Extraocular Muscles ◽  
Axonal Membrane ◽  
Electron Microscope Autoradiography ◽  
Diisopropyl Fluorophosphate ◽  
Speed Of Response ◽  
Microscope Autoradiography

The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n average of 6.4 +/- 2.1 X 10(7) DFP-binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP-binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.

Incorporation of Cholesterol into Peripheral Nerve Myelin

1972 ◽  
Vol 30 ◽  
pp. 334-335
Author(s):  
Frank A. Rawlins
Keyword(s):  
Electron Microscope ◽  
Sciatic Nerve ◽  
Electron Microscope Autoradiography ◽  
Myelin Sheaths ◽  
Microscope Autoradiography ◽  
Grain Distribution ◽  
Sciatic Nerves ◽  
Autoradiographic Analysis ◽  
Old Mice ◽  
Short Times

Several speculations exist as to the site of incorporation of preformed molecules into myelin. The possibility that an autoradiographic analysis of cholesterol-1,2-H3 incorporation at very short times after injection might shed some light in the solution of that problem led to the present experiment.Cholesterol-1,2-H3 was injected intraperitoneally into 24 tenday old mice. The animals were then sacrificed at 10,20,30,40,60,90,120 and 180 min after the injection and the sciatic nerves were processed for electron microscope autoradiography. To analyze the grain distribution in the autoradiograms of cross and longitudinal sections from each sciatic nerve myelin sheaths were subdivided into three compartments named: outer 1/3, middle 1/3 and inner 1/3 compartments.It was found that twenty min. after the injection of cholesterol -1.2-H3 (Figs. 1 and 2), 55% of the total number of grains (t.n.g) found in myelin were within the outer 1/3 compartment, 9% were within the middle 1/3 and 36% within the inner 1/3 compartment

Synthesis and Localization of Sulphated Mucopolysaccharide in Megakaryocytes and Platelets of the Rat, An Analysis by Electron-Microscope Autoradiography

1972 ◽  
Vol 10 (3) ◽  
pp. 705-717
Author(s):  
G. G. MacPHERSON
Keyword(s):  
Electron Microscope ◽  
Golgi Apparatus ◽  
Blood Platelets ◽  
Total Activity ◽  
Electron Microscope Autoradiography ◽  
Dense Granules ◽  
Level Of Activity ◽  
Microscope Autoradiography ◽  
Almost All

Electron-microscope autoradiography has been used to investigate the synthesis and localization of sulphated mucopolysaccharide in megakaryocytes and blood platelets. Following 10-min incubation of bone marrow with 35S-sulpahte in vitro the majority of the activity in megakaryocytes was associated with the Golgi apparatus, but a substantial proportion was associated with other cytoplasmic organelles, suggesting either rapid transport or sulphation of mucopolysaccharide outside the Golgi apparatus. Three hours after the intravenous injection of 35SO4 only a small proportion of the total activity was associated with the Golgi apparatus, most being associated with demarcation membranes and dense granules, while 12 h after injection almost all the activity was associated with demarcation membranes and granules. A rising proportion of activity localized solely on the demarcation membranes suggested that they may possess some activity of their own. Autoradiographs of blood platelets prepared 72 h after the injection of 35SO4 were analysed. It was shown that most of the activity was associated with the α-granules, but there was strong evidence that the platelet membrane possessed a low level of activity.

Timing of nucleolar DNA replication in Amoeba proteus

1976 ◽  
Vol 22 (3) ◽  
pp. 521-530
Author(s):  
I. Minassian ◽  
L.G. Bell
Keyword(s):  
Electron Microscope ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Central Region ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Amoeba Proteus ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
G2 Phase

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.

Studies on the Synthesis and Composition of Extracellular Mucilage in the Unicellular Red Alga Rhodella

1974 ◽  
Vol 16 (1) ◽  
pp. 1-21
Author(s):  
L. V. EVANS ◽  
MAUREEN E. CALLOW ◽  
ELIZABETH PERCIVAL ◽  
V. FAREED
Keyword(s):  
Electron Microscope ◽  
Uronic Acid ◽  
Red Alga ◽  
Electron Microscope Autoradiography ◽  
Golgi Bodies ◽  
Microscope Autoradiography ◽  
Reducing Substances

35SO42- has been used to investigate the production of extracellular mucilage by log-phase cells. Uptake of isotope occurs most rapidly in the light, when cells are actively dividing. The mucilage comprises about 50% carbohydrate, 16% protein and 10% sulphate. The major sugar is xylose; uronic acid, a small amount of galactose, glucose (trace) and 2 reducing substances are also present. Methylation studies have established the major linkages. Electron-microscope autoradiography shows that the mucilage is packaged in the Golgi bodies, passing to the plasmalemma in large vesicles. Sulphation of the mucilage occurs in the Golgi cisternae.

scholarly journals Resolution of electron microscope autoradiography. IV. Application to analysis of autoradiographs.

1978 ◽  
Vol 76 (1) ◽  
pp. 127-145 ◽  
Author(s):  
M M Salpeter ◽  
F A McHenry ◽  
E E Salpeter
Keyword(s):  
Electron Microscope ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Cross Scatter ◽  
Best Fit ◽  
Subsequent Comparison ◽  
Complex Cases

The previous publications of this series described the expected grain distributions around model radioactive structures in EM autoradiographs as a function of the specimen resolution. This family of expected distributions was called the "universal curves". In the present study, experiments on 14C-sources were compared, significant differences were found depending on the energy of the isotope. These differences were primarily in the tails of the distributions, and are therefore important in correcting for cross-scatter when analyzing electron microscope autoradiographs. Using the universal curves unique for 125I, 3H, and 14C, we designed three sets of transparent overlays, or "masks", one set for each of these isotopes. The masks can be used by an investigator in a manner similar to that suggested by Blackett and Parry to generate grain distributions in autoradiographs on the basis of any desired hypothesis regarding the levels of radioactivity in different structures. A subsequent comparison between these generated distributions and those obtained from the observed grains in these autoradiographs leads to a determination of the most likely levels of radioactivity in the tissue. A computer (described in an Appendix by Land and Salpeter) can be used to find the "best fit" levels of radioactivity in complex cases. The accuracy of the masks was checked on generated line sources for each of the three isotopes.

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