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Primitivfibrille, oft wohl bereits Elementarfibrille, an einer bestimmten Stelle meiner Präparate zu verfolgen im Stande bin."

In 1877 GERLACH described the nerve endings of the frog as branching dichotomously to form an intravaginal network which runs through the contractile substance of the muscle fiber in close relation to, if not in actual continuity with, the contractile fibrillae. He appears to have been influenced in his deductions by the fact that in staining with gold chloride, he obtained reactions from certain elements of the sarcous substance which were similar to those obtained in nerve endings. In 1889 he repeated these investigations with methylene blue, and claimed that this method substantiated his previous views. Subsequent research by others has not confirmed his results nor upheld his deductions; nor can his own drawings be said to support the claims he attempted to establish. To one conversant with intra-vitam staining with methylene blue, it will appear a very doubtful procedure to base results on the similarity of staining reactions. By this intra-vitam method one constantly sees fine blue-stained fibers in the intermuscular connective tissue which, taken alone, might simulate nerve fibrillae. At times one observes beautifully stained examples of myofibrillae either as very fine closely-arranged points or as continuous structures. On this ground alone, therefore, it is obvious that it would not be possible to deduce a nervous plexus either within or without the muscle-cell. In addition, there must be present an undoubted continuity of structure with an unmistakable nerve fiber, together with any characteristic appearance which one is accustomed to find in corresponding terminals.

In 1896 APÁTHY drew attention to the manner in which the axis cylinder divides in invertebrate muscle fibers. He describes the primitive fiber as entering the muscle cell and there dividing into fine fibrillae (primary fibrils). These, however, do not form a plexus but either send comparatively thick branches (secondary fibrils) to adjacent muscle-cells there to form terminations from which it may be that further branches

| Mitheil. a. d. Zoolog. Station zu Neapel., Bd. XII, 1896, p. 505.

(tertiary fibrils) may proceed to other muscle cells; or they divide repeatedly into ever finer fibrils till ultimately perhaps, elementary fibrillae result. He often saw very fine nerve fibrillae pass out of a muscle cell into the neighboring connective tissue and it might be, branch there. As to the ultimate fate of the finest fibrillae he is uncertain, but he declares : "Es kommt mir an wahrscheinlichsten vor, das sie mit anderen ähnlichen ein intermuskulares Elementargitter bilden.”1

The results of APÁTHY led RUFFINI in 1900 to re-examine nerve endings in the human muscle which he had stained by his chloride of gold method. Here he found in some of his specimens fine fibers which he had not previously noted and which he now described under the name of ultra-terminal fibrillae. These are very fine, non-medullated fibrillae which pass out from the motor end-plate. "This fibrilla" (ultraterminal), he says, "after an unbranched course, more or less long, may terminate in the same muscular fiber on which rests the plate from which it is derived, or, as is often the case, put itself into relation with one of the neighboring striated fibers. From it collaterals may at times come off." It may end in a swelling or in a second small plate (plaquette); in one case from a plaquette another fibre was seen to pass out, but its course could not be followed. Summing up the results of his examination, he says: "The motor plates in man do not represent the true and real terminations of motor nerves, because beyond the plate there exists a well demonstrable anatomical continuity shown by non-myelinated nerve fibrillae of which we do not know the last relations." This is obviously a statement very closely akin to that given by APÁTHY in the summing up of his results.

2

Later PERONCITO, working with RUFFINI and using his gold chloride method, describes in Lacerta muralis and Lacerta viridis the ultraterminal fibrilla as separating itself from the

1 Ibid., p. 695.

2 RUFFINI: Sulle fibrille nervose ultraterminale nelle piastre motrici dell' uomo. Rivista di pat. nerv. e ment., 1900, V. 5.

nerve fiber at the point where it loses its medullary sheath and penetrates the motor plate ; from here it may pass to end on a muscle fiber or in a neuromuscular spindle.

In any discussion on nerve endings it is well to recognize the facts that are universally accepted. Thus it is well established that a motor nerve may branch repeatedly before ending, or to put it otherwise, the peripheral ending of a motor neurone is connected with many muscle fibers. This branching occurs at a node. Each branch is a medullated fiber smaller in calibre than the parent stem and it ultimately loses the medullary sheath and breaks up to form a nerve ending usually on one muscle fiber, to which it alone is attached.

To this very general statement there are a variety of exceptions. Thus, two or more endings may go to one muscle fiber; or a non-medullated nerve may pass off from a node; occasionally, though more rarely, a non-medullated nerve may be seen leaving a medullated nerve where no node is apparent. To define concisely and yet accurately the term motor nerve ending, either from an anatomical or physiological standpoint, is in the present state of our knowledge by no means easy. For our present purpose a motor ending may be regarded as that peripheral part of the nerve which, on reaching a muscle fiber, loses its medullary sheath and breaks up into more or less numerous non-medullated' branches or end-twigs which enter into a more or less close relation to the muscle fiber. Having thus defined a motor ending, the significance of the term ultraterminal fibrilla is the better understood; this is a fine non-medullated fibril which passes from one of the twigs of the nerve termination to a region beyond the primary ending. Here it enters into relation with the muscle fiber on which rests the ending from which it originally sprangor with some adjacent muscle fiber or with a neighboring muscle spindle.

1

Dogiel has described a medullary sheath as occurring in the nerve end ing (Archiv f. mikr. Anat., Bonn, 1890, p. 314). This I have never seen, though the dye oozing from the axis cylinder at times gives a resemblance to such.

The following investigations were undertaken by me to determine how the motor nerve endings on the muscles of the frog are related to other structures, and also to ascertain, as far as possible, how the terminal nerve fibrils finally break up and disappear. Especially did it appear necessary to compare the results observed by RUFFINI and others using gold chloride methods with the results obtained by the intra-vitam methylene blue method.

Method: The muscles of the frog used were either the M. sartorius, the M. peroneus or the M. tibialis anticus. Into these was injected with a hypodermic syringe, a very weak solution of methylene blue in various salt solutions, for this research has been carried on as a preliminary part of an investigation on the effect of various salts and poisons on motor nerve endings. Grammolecular solutions of the following salts, among others, were used : sodium chloride, sodium carbonate, sodium ammonium phosphate, magnesium sulphate. Nerve endings can be obtained by a solution of methylene blue in distilled water, or with methylene blue in solution with any of the above salts; but the most constant and best results are obtained if sodium chloride is present in the solution.

So far as the present paper is concerned, the following solution was constantly employed:

Methylene blue (GRÖBLER's nach EHRLICH) 0.5% sol.
Sodium chloride solution, 0.58% sol.
Aqua destil.

17 cc. This was found to be the most suitable strength of methylene blue to use, though one of half this strength often answers well, especially for sensory endings and sympathetic plexures on blood vessels. The largest and most complex endings were seen when the muscle was injected with the above solution after there had been added to it a few drops of a weak alkaline salt, such as sodium ammonium phosphate, and then a very weak faradic current passed through the nerve trunk for a few seconds.

A few minutes after injecting the solution the muscle is cut out and exposed on a glass slide which has been moistened

I or 2 CC.

2 CC.

with normal salt solution. Within a varying time, usually about five minutes, the nerve endings begin to appear. As soon as these are well marked the muscle is fastened to cork and placed in ammonium molybdate solution (5%) at a temperature near freezing point. Temperature plays a very important part in the subsequent treatment of the tissues. If it rise at any point of the procedure—e. g. when washing in water or passing through alcohol—the methylene blue dissolves out from the very fine fibrillae. If the tissue has to kept over night in molybdate it is well to place it, especially in summer, in a refrigerator. When passing through alcohol, the vessel should be surrounded by ice-cold water.

The addition of HCl to the molybdate solution is not necessary; I have found it even a disadvantage for the finer results. BETHE in his recent work has abandoned its use.

After removal from the molybdate, the muscle, if too thick, is cut in a freezing microtome, examined in water, nd only that part kept which shows traces of nerve endings. The tissue is now passed through 95% and absolute alcohol, preferably, as stated above, at a low temperature; then from xylol into paraffin. It is important that it remain sufficiently long in xylol to remove the alcohol; otherwise the temperature of the melted paraffin will cause any alcohol present to remove the methylene blue from the fine fibrillae. It remains in paraffin for two hours. The thickness of the section varies with the object aimed at. To get long stretches of nerve endings, it is best to cut from 20 to 50 micra; if one wish to study the relation of the endings to the muscle cell, a thickness of from 5 to 10 micra must be used.

After numerous experiments with various dyes as a counterstain, the following was found most suitable, inasmuch as it dyed the muscle fiber an orange, the sheath of HENLE a rosepink and the neurilemma a faint pink:

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Acid fuchsin
Orange “G”
Alcohol, absolute,
Aqua destil.

60 cc.

240 cc.

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