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.

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.

Quantitation of Fluorophosphate - Reactive Esterase Sites in Rat Liver Using Electron Microscope Autoradiography

1975 ◽  
Vol 33 ◽  
pp. 466-467
Author(s):  
G. C. Budd
Keyword(s):  
Electron Microscope ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Average Concentration ◽  
Electron Microscope Autoradiography ◽  
Cytoplasmic Granules ◽  
Diisopropyl Fluorophosphate ◽  
Microscope Autoradiography ◽  
Cytoplasmic Structures ◽  
Silver Grains

Following the application of 10-4 molar tritium labeled diisopropyl fluorophosphate (3H-DFP) to fresh or glutaraldehyde fixed rat liver, fluorophosphate-reactive (FPR) sites within the hepatocytes were measured using quantitative electron microscope autoradiography (EMARG). Based on the distribution of autoradiographic silver grains, most of the FPR sites were concentrated in the rough and smooth endoplasmic reticulum and associated ground cytoplasm. Cytoplasmic granules, comprising autophagic and residual granules also contained FPR sites. The average concentration of sites within each of these cytoplasmic structures was obtained from combined morphometric and grain density analyses of the EMARGs and light microscope sections of the same epoxy embedded liver specimens.

scholarly journals SENSITIVITY IN ELECTRON MICROSCOPE AUTORADIOGRAPHY I. THE EFFECT OF RADIATION DOSE

1972 ◽  
Vol 20 (6) ◽  
pp. 425-434 ◽  
Author(s):  
MIRIAM M. SALPETER ◽  
MARIA SZABO
Keyword(s):  
Ascorbic Acid ◽  
Electron Microscope ◽  
Radiation Dose ◽  
Surface Area ◽  
Test Specimen ◽  
Dose Dependence ◽  
Unit Surface Area ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Unit Surface

Sensitivity in electron microscope autoradiography using Ilford L4 emulsion was shown to be affected by radiation dose ( i.e., number of decays in test specimen per unit surface area). The sensitivity tended to be higher with lower doses. This dose dependence was most marked with Microdol X and least with gold latensification-Elon ascorbic acid development. Possible consequences for quantitation in electron microscope autoradiography are discussed.

scholarly journals QUANTITATIVE ASSAY OF ESTERASES IN END PLATES OF MOUSE DIAPHRAGM BY ELECTRON MICROSCOPE AUTORADIOGRAPHY

1972 ◽  
Vol 20 (12) ◽  
pp. 1059-1068 ◽  
Author(s):  
MIRIAM M. SALPETER ◽  
HELMUT PLATTNER ◽  
ANDREW W. ROGERS
Keyword(s):  
Electron Microscope ◽  
Predominantly White ◽  
Synaptic Cleft ◽  
Postsynaptic Membrane ◽  
Electron Microscope Autoradiography ◽  
Diisopropyl Fluorophosphate ◽  
Mouse Diaphragm ◽  
Microscope Autoradiography ◽  
Alternative Hypotheses ◽  
Motor End Plates

The inhibitor diisopropyl fluorophosphate (DFP) reacts covalently with a number of enzymes including acetylcholinesterase (AChE). The compound pyridine-2-aldoxime methiodide reactivates phosphorylated AChE faster than the other DFP-sensitive sites. Mouse diaphragm was incubated with 3H-DFP, and the radioactivity in the motor end plates was assessed by electron microscope autoradiography. Both the sites which are rapidly reactivated and those which are slowly reactivated by pyridine-2-aldoxime methiodide were evaluated. The distribution of silver grains was compatible with several alternative hypotheses, i.e., that both AChE and other DFP-sensitive enzymes are located on or around the postsynaptic membrane alone, are distributed uniformly throughout the synaptic cleft and are associated equally with both pre- and postsynaptic membranes. This distribution of developed grains was identical with that found in the larger end plates of mouse sternomastoid. The values for the number of molecules of 3H-DFP, whether expressed in terms of square micra of junctional membrane or of cubic micra of synaptic cleft, were also practically identical with those found for sternomastoid. These similarities between the diaphragm, a red muscle, and the sternomastoid, which is predominantly white, suggest that there may be a constant relationship between the sites that bind DFP and the unit dimensions of the subneural compartments concerned.

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

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