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 sprang, or 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. 1 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 (GRÜBLER'S nach EHRLICH) 0.5% sol. I or 2 cc. Aqua destil. 2 CC. 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 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, and 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 : In the frog's muscle the nerve ending has no ground plate in which the branches ramify. The ramifications are not localized but are spread over a relatively large and apparently variable area of the muscle fiber. Usually but one medullated nerve ends in a muscle fiber, though two medullated nerves may be seen at times and three have been described; occasionally two medullated nerve fibers and one non-medullated and much finer fiber may go to the muscle fiber. When more than one nerve goes to a muscle fiber it is often possible to trace the origin of these to the same nerve stem. In no case was there even the suggestion of one of these fibers coming from a nerve whose course lay distinctly apart from the others. If more than one nerve go to the muscle fiber, the places where the nerves enter into contact with the fiber are, if not always, at least most frequently, in close proximity to one another. In short, on the muscle fiber the area to which the entering nerves apply themselves relative to the entire length of the muscle fiber is limited. Several varieties of motor endings have been described. Thus, there are the four or five types of CUCCATI which RETZIUS would reduce to two; the one exemplified by the branching plate, the other by the broad band. At present, any classification must be but temporary; with equal justification several varieties may be classed as typical by one and rejected by another. For example, one might well wish to add to the types of RETZIUS that more strictly localized variety which DOGIEL has described and which occurs not unfrequently in certain muscles—a variety which closely resembles the branching of mammalian nerve endings only without an end plate.1 There is, however, one type which is generally recognized as predominating-das Stangen-geweih of KÜHNE. In it the nerve after losing its medullary sheath divides more or less dichotomously and spreads itself along the length of the muscle fiber. The band variety is less common; one notes that the better stained the preparation the less frequently it appears. 1 Arch. f. mikrosk. Anat., 1890, Bd. xxxv. In the frog the end-arborizations do not terminate necessarily on one muscle fiber. It is not unusual to find that, while the majority of the terminal fibers confine themselves to one muscle fiber and to a certain definite area, one or more fine non-medullated fibrillae pass far beyond the area or to a neighboring muscle fiber. So common is this that in well stained preparations one is ever expectant of finding at least traces of such fibrillae. They are often difficult to observe; but the cause of this is not so much that they do not readily stain, as that it is hard to fix the dye and easy to have it extracted during the stages subsequent to fixation. Such non-medullated fibrillae may be termed ultraterminal. A convenient way to describe them is to classify them according to the manner in which they end, so far, at least, as such can be traced at present. With this in view, we might describe them as follows: (1) Relatively thick, non-medullated fibers which pass to adjacent (3) Very fine non-medulated fibrillae which detach themselves at a) by getting so faint and so fine that it becomes impossible to follow them farther (Fig. 1; Fig. 2; Fig. 3). b) by terminating at what appears as a much thickened knob (similar to Fig. 1, B and C) c) by breaking up into a plexus from which some at least of the fibrillae disappear in the muscle fiber while others continue on (Fig. 2) d) by forming a plexus which enters into close relationship with a typical nerve ending (Fig. 3) e) by breaking up after a relatively long course to form a small localized ending, each termination of which is furnished with a knob (Fig. 6). |