scholarly journals ELECTRON MICROSCOPE RADIOAUTOGRAPHY AS A QUANTITATIVE TOOL IN ENZYME CYTOCHEMISTRY

1967 ◽  
Vol 32 (2) ◽  
pp. 379-389 ◽  
Author(s):  
Miriam M. Salpeter

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.

The Copley Medal is awarded to Professor B. Katz, F. R. S. Professor Katz’s researches have mainly been concerned with the mechanism of junctional transmission between nerve and muscle, but earlier he played an important part in helping Hodgkin and Huxley to establish the ionic theory of the nervous impulse. He set out to examine the nature of the end-plate potential with intracellular micro-electrodes, and showed that the arrival of a nerve impulse at the motor end-plate had the effect of short-circuiting the resistive membrane of the muscle fibre so as to set up a propagated action potential in the fibre. This led him to the discovery of the spontaneous ‘miniature’ end-plate potentials, which are due to a quanta! random release from the nerve endings of the chemical transmitter acetyl­choline. He went on to develop as a precise and quantitative tool the technique of applying minute quantities of drugs at selected spots by releasing them electro-phoretically from micropipettes, in order to study the pharmacology of the motor end-plate. The concepts and methods introduced by Professor Katz in each phase of this work have been widely applied to study junctional transmission elsewhere, for example in the brain and spinal cord, so that he has had a considerable in­fluence on the development of our ideas about the detailed working of the nervous system.


The Copley Medal is awarded to Professor B. Katz, F. R. S. Professor Katz’s researches have mainly been concerned with the mechanism of junctional transmission between nerve and muscle, but earlier he played an important part in helping Hodgkin and Huxley to establish the ionic theory of the nervous impulse. He set out to examine the nature of the end-plate potential with intracellular micro-electrodes, and showed that the arrival of a nerve impulse at the motor end-plate had the effect of short-circuiting the resistive membrane of the muscle fibre so as to set up a propagated action potential in the fibre. This led him to the discovery of the spontaneous ‘miniature’ end-plate potentials, which are due to a quantal random release from the nerve endings of the chemical transmitter acetylcholine. He went on to develop as a precise and quantitative tool the technique of applying minute quantities of drugs at selected spots by releasing them electrophoretically from micropipettes, in order to study the pharmacology of the motor end-plate. The concepts and methods introduced by Professor Katz in each phase of this work have been widely applied to study junctional transmission elsewhere, for example in the brain and spinal cord, so that he has had a considerable influence on the development of our ideas about the detailed working of the nervous system.


1973 ◽  
Vol 21 (7) ◽  
pp. 634-652 ◽  
Author(s):  
VIRGINIA M. TENNYSON ◽  
LEON T. KREMZNER ◽  
MIRO BRZIN

Acetylcholinesterase (AChE) activity has been studied in the myotube and in extrajunctional skeletal muscle of the rabbit fetus. Some observations on the developing motor end plate are included. Both insoluble and soluble AChE's are present at all stages, but butyrylcholinesterase seems to be absent. Enzymic activity is high during myotube formation. The insoluble AChE probably corresponds to the cytochemical end product which is bound to the elements of the reticulum in the myotube, i.e., the nuclear envelope, the sarcoplasmic reticulum and some elements of the Golgi complex. The possibility that the enzyme-containing reticulum is involved in the spontaneous contraction of early myotubes is discussed. The number of active sites of reticulum-bound enzyme decreases markedly as the myotubes mature into early muscle. The soluble AChE is probably derived from the AChE-containing mononuclear cell and the Schwann cell which accompanies the spinal nerve to the motor end plate. These cells have a random distribution of end product and are probably the source of a similar end product found at the surfaces of these cells and at the sarcolemma of adjacent myotubes. The following possibilities that are discussed are that soluble AChE may (a) play a role in fusion of myotubes, (b) be involved in widespread sarcolemmal acetylcholine sensitivity and (c) contribute to the junctional enzyme. The randomly distributed end product in the AChE-containing mononuclear cell permits the tracing of its further differentiation. This cell appears to be a stem cell, which can fuse directly with the myotube; it can give rise to a more differentiated type of myoblast, as well as to the muscle satellite cell. AChE-containing mononuclear cells may also contribute to the cells associated with the neuromuscular junction. The soluble AChE in the muscle satellite cell seems to be associated with an "active" phase of this cell, since enzymic activity is not seen in the adult stage in which it is presumed to be dormant.


1969 ◽  
Vol 42 (1) ◽  
pp. 122-134 ◽  
Author(s):  
M. M. Salpeter

The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP-3H. Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per µ3 of cleft tissue or 12,000 sites per µ2 of postjunctional membrane surface area. A considerable concentration is also present in the teloglial cap. It has previously been demonstrated (Rogers et al., 1966) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)—a compound which selectively reactivates phosphorylated acetylcholinesterase. In the present study, it was found that this ratio of 1:2 holds also on a fine-structural level. Muscle mast cells were found to have a heavy concentration of bound DFP.


1988 ◽  
Vol 106 (6) ◽  
pp. 2087-2093 ◽  
Author(s):  
M M Salpeter ◽  
M Marchaterre ◽  
R Harris

A scanning electron microscope (SEM) autoradiographic technique was calibrated and used to determine the site density of acetylcholine receptors within 250 micron of the neuromuscular junction in innervated as well as 3- and 10-d denervated sternomastoid muscle of the mouse. In all these groups sharp gradients of receptor site density are seen around the endplates in the first 2-7 micron, continuing less sharply to between 25 and 50 micron. Beyond 50 micron (to 250 micron) a spatial density gradient is present 3 d after denervation, but none exist by 10 d. These results suggest that the postdenervation steady-state extrajunctional receptor site density is reached sooner near the junction than away from the junction. The usefulness of SEM autoradiography to study the expression and distribution of membrane molecules at high resolution is demonstrated.


1995 ◽  
Vol 132 (2) ◽  
pp. 97-104
Author(s):  
Naoko Tetsuo ◽  
Mitsuhiro Tsujihata ◽  
Akira Satoh ◽  
Toshiro Yoshimura ◽  
Tatsufumi Nakamura ◽  
...  

1963 ◽  
Vol 157 (969) ◽  
pp. 536-561 ◽  

Resting potentials, action potentials, and miniature end-plate potentials have been re­corded from isolated phrenic-diaphragm preparations of the rat during and after irradiation with X-rays. Relatively small doses of a few thousand roentgens have no obvious effect on the preparation for many hours but larger doses, of the order of 70 to 150 kr irreversibly block neuromuscular transmission. The block is not accompanied by any change in the size of action potentials, resting potentials, membrane constants or miniature potentials recorded in the muscle with intracellular electrodes, or in the size of action potentials recorded in the nerve. Records made at the motor end-plate show that the cause of the block is a ‘pre-synaptic ’ failure of impulse propagation in the intramuscular part of the nerve. The time course of the failure depends largely on the rate at which X-rays are delivered to the pre­paration: at a high dose-rate (70kr/min) the block develops rapidly and is accompanied by an increase in the frequency of miniature potentials; at a low dose-rate (7 kr/min) larger doses are required, the latency is longer and the miniature potentials continue at a normal frequency. The sequence in which different parts of the muscle become blocked, the abrupt nature of the failure at an individual motor end-plate, and the increase in frequency of the miniature potentials together suggest that the action of X-rays is to block conduction in the nerve near its terminals, possibly by depolarizing points where the axons branch and the safety factor for the propagation of impulses is low. The results reported in this paper do not support the hypotheses that small doses of X-rays at a high or a low dose-rate lead to an initial 'enhancement' of function or that they produce immediate and reversible changes in the permeability of excitable membranes to ions.


2003 ◽  
Vol 27 (4) ◽  
pp. 426-434 ◽  
Author(s):  
Frank Spaans ◽  
Jan-Willem Vredeveld ◽  
Humphrey H.E. Morré ◽  
Bart C. Jacobs ◽  
Marc H. De Baets

1981 ◽  
Vol 51 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Ryuji Hazama ◽  
Mitsuhiro Tsujihata ◽  
Masataka Mori ◽  
Masaharu Takamori ◽  
Kazutake Mori ◽  
...  

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