A PECULIAR STRUCTURE IN THE ELECTROPLAX OF THE , GUTTATUS JAMES G. HUGHES, JR. From the Histological Laboratory of Princeton University, U. S. A.

THREE FIGURES The purpose of this paper is to determine the function and composition of the peculiar pointed fibers and long pointed rods lying in the electric layer of the electroplaxes of the stargazer, . Before proceeding with a discussion of these rods, a brief description of the electric organ of this (according to Dahl- gren)' will be given. The electric apparatus is composed of two organs, which form two vertical columns roughly oval in horizontal section, and placed behind and somewhat under each eye. Each organ extends from the peculiar bare spot on the top of the head down to the tissues which form the roof of the oval cavity; and is composed of about 200 thin layers of electric tissue, which extend horizontally all the way across the organ. These layers of tissue are flat, and always at the same distance from one another. Each layer contains about 20 electroplaxes, the outlines of which present a very irregular or scalloped appear- ance. The electric tissue in which the electroplaxes are im- bedded is in appearance a jelly-like or mucous-like tissue, usu- ally known as electric connective tissue, and which I have shown in the course of my work to be of the same composition as white fibrous connective tissue. The nerve and blood supply runs in the above tissue. The general form of a vertical section of an electroplax is shown by figure 1, whioh is a drawing of part of a section of a single electroplax. Each electroplax is composed of three principal layers, a nervous or electric layer which forms the upper surface, a

1 Anat. Am., Bd. 29, S. 387, 1906. 97 98 JAMES G. HUGHES, JR. middle layer, and a lower or nutritive layer which along with the middle layer is evaginated into a large number of long papillae. All three layers are deeply marked with a dense series of fine striations, which are peculiar to the electroplaxes of several other . The upper or electric surface is flat and smooth and receives the nerve endings. The current of electricity runs downward through the organ which produces it, and thus the nerve endings in accordance with Pacini’s law are found on the negative pole of the electroplax. Proceeding directly to the subject of this paper, we may say that one of the most interesting of the points noted in the elec- troplaxes, when properly fixed and stained with iron hematoxy- lin, is a series of rod-like or thread-like objects running hori- zontally in the electric layer, among, above and below the nuclei and without any apparent connection with them (figs. 1 and 2).2 These rods are of various sizes and shapes, and in form are said to resemble the classic thunderbolts seen in the hand of rep- resentations of Jove. They usually taper slowly end branch extensively at one or both ends. Some of these brznches some- times seem to be mere lines, while others are wide and heavily pointed; at their other ends the rods are usually rounded; this latter appearance may be due, however, to the cut ends of the rods, for as noted above they sometimes branch at both ends. Some are short and heavy in appearance while others me long and thread-like. Peculiar looping, twisting, or knot-like bends are sometimes found at points on the longer rods. The out- lines and contour of these rods are always smooth. Their size may vary from thick or thin rods of over 300 1.1 in length down to small ones that do not exceed 1 p. In those electroplaxes where the rods are few they sometimes lie parallel and point in a definite parallel direction, while in others where the rods are very numerous they do not seem to have any definite arrangement. In this latter condition the rods present a very wavy appearance. Their form may be seen in figures 1 and 2, which are drawings of the electric layer of an electroplax when All the figures are drawings of sections of electroplaxes of Astroscopus guttatus. 99

Fig. 1 Vertical section of part of an electroplax. The whole electroplax as well as the electric connective tissue is shown; rods arc sccn lying in the electric layer. X 1200. 100 JAMES G. HUGHES, JR.

Fig. 2 Horizontal section through an electroplax. Only the electric layer is seen; a, an extensively branching rod; b, fine branches of the above rod; c, x large characteristic loop io a rod;d, a cut end of a rod; e, nuclei of the electric layer. X 1200. stained with iron hematoxylin. These drawings show the electric layer in which the rods are found in horizontal section (fig. 2) and in vertical section (fig. 1). The purpose, function, and chemical composition of these rods have been previously unknown to histologists. In order to determine anything in respect to their function or purpose, a knowledge as to the class of organic substance to which they belong, whether muscle connective tissue nervous or chitinous ELECTROPLAX OF ASTROSCOPUS 101 and also a rough knowledge of their chemical composition is imperative. The contour and form of these rods as they appear under the microscope resemble both smooth muscle fibers and fibers of elastic connective tissue. The belief that the function of these rods was somewhat of the nature of support for the delicate substance of the electroplax, and the fact that their form resembled connective tissue fibers led the writer to take for one of his first hypotheses, that they were of some form of connective tissue, and to perform accordingly the following series of experiments. As the most logical and best way for determining the kind of connective tissue, if any, of which the rods might be composed, a number of stains used by other investigators to identify similar substances were applied and the results noted. Controls were used on known tissues. Before taking up the connective tissue stains, however, a description of the results from the iron hematoxylin staining is now noteworthy; the material used being fixed in pure corrosive sublimate. The jelly connective tissue stained a very light gray. The nutritive, striated, and electric layers stained a much darker gray. The nuclei in all the layers stained somewhat black. The pointed fibers, and rods found in the electric layer stained a deep black, thus being clearly differentiated from the surround- ing cytoplasm. In some electroplaxes they were very numer- ous, while in others the number was rather small. The connective tissue stains applied as follows: (1) Mallory’s connective tissue stain, using the modifica- tion given in Lewis’ “Text-book of histology.” White fibrous connective tissue should stain blue in this medium. Paraffin sections of the electroplaxes fixed in corrosive subli- mate were stained for 10 to 12 minutes in a 1 per cent aqueous solution of acid fuchsin. They were then transferred directly to a stain consisting of 0.05 grain of aniline blue (soluble in water) and 0.2 grain of orange G dissolved in 100 cc. of a 110 per cent aqueous solution of phospho-molybdic acid. In this they remained from 2 to 3 minutes. They were then rinsed in distilled water, dehydrated rapidly, cleared and mounted. 102 JAMES G. HUGHES, JR.

A description of an electroplax as seen under the 2 mm. oil immersion lens is as follows: The white fibrous or electric con- nective tissue stained a light blue or purple. The electric nutri- tive and middle layers or the electroplax proper stained a red- dish purple, and the nuclei as a whole in all of the layers, stained somewhat lighter than their surrounding cytoplasm. The peculiar rods and fibers stained a deep red, and were thus clearly differentiated from the other elements of the elec- troplax. They were very numerous and as noted above their outlines were always smooth. Blood corpuscles lying in the jelly connective tissue, of which there were only a few, stained a brilliant red, much the same as the rods. The white fibrous or jelly electric connective tissue (be- tween the electroplaxes), as noted above, stained a light blue. The pointed fibers and rods stained a deep red. This would indicate therefore, that these rods are not composed of white fibrous connective tissue. It would seem also that they are not muscle for they stained a different shade of color from the rest of the electroplaxes, which I have found in the course of my work to stain much the same as muscle. (2) Van Gieson’s connective tissue stain, in which white fibrous connective tissue should stain red: Pzraffin sections of the electroplaxes fixed in corrosive sub- limate were stained for 4 to 5 minutes in a 1 per cent aqueous solution of hematoxylin. They were rinsed in distilled water and transferred to a stain consisting of a saturated aqueous solution of picric acid containing 20 per cent acid-fuchsin. They remained in here 15 to 20 minutes and were then rinsed, cleared and mounted. The white fibrous connective tissue layer stained pink. The three layers of the electroplax stained brown. The nuclei and rods stained the same color, that is, brown; and, while the nuclei could be seen with difficulty, the rods were scarcely visible owing probably to their similar refractive in- dex. The fact therefore that the rods did not stain the same color as the white fibrous connective tissue, which stained pink, indicates again that they are not composed of white fibrous connective tissue. By Van Gieson’s stain, therefore, I have ELECTROPLAX OF ASTROSCOPUS 103 confirmed the evidence as presented by Mallory’s stain in re- spect to the composition of the rods; that is, they are not white fibrous connective tissue. I now undertook to apply stains which were tests for elastic fibers, and the first under this head is Weigert’s resorcin-fuchsin stain in which connective tissue according to Weigert and other writers stains dark blue. The stain was made up as follows: One per’ cent of basic fuchsin and 2 per cent of resorcin were dissolved in water; 50 cc. of the solution were raised to the boiling point, and 25 cc. of liquor ferri sesquichlorate P.G. were added and the whole boiled with stirring from 3 to 5 min- utes; a precipitate was formed. After cooling, the liquid was filtered, and the precipitate which remained on the filter was boiled with 50 CC. of 95 per cent alcohol. It was then a.llowed to cool, filtered and the filtrate made up to 50 cc. with alcohol, and 1 cc. of hydrochloric acid added. Paraffin sections of the electroplaxes fixed in corrosive sub- limate were stained for 6 hours in the above alcoholic solution of the precipitate. They were then washed in 95 per cent alcohol, dehydrated quickly, cleared and mounted. -1 descrir- tion of the results are as follows: The electroplax proper, with its three layers, did not take the stain at all. The jelly electric tissue around the electroplax stained a brilliant blue. The rods contained in the electric layer of the electroplax were invisible. The fact therefore that the rods did not take the stain at all shows they are not composed of elastic connective tissue. The control used in this case for elastic tissue was a section of the ligamentum nuchae of a horse, which, when put in the stain for exactly the same time as the electroplax, came out a deep blue. The ligamenturn nuchae was chosen as a con- trol since it is, perhaps, the best known and best representative of elastic tissue found in the kingdom. Not desiring to rely solely on tnt. above stain to prove that the rods are not composed of elastic tissue, the electroplaxes were treated with the digesting fluid, pepsin, and the results noted. Vertical sections of electroplaxes, having been freed from paraffin, were put for 3 minutes in a very weak solution 104 JAMES G. HUGHES, JR. of pepsin in 0.2 per cent HCL (the concentration being 0.5 gram of commercial pepsin to 100 cc. of 0.2 per cent aqueous HCL). The electroplaxes were then treated with the following stains: (1) Iron hematoxylin. The result was that all the rods and part of the nuclei, and less dense portions of the electroplax had been digested. (2) .Mallory’s connective tissue stain was applied, the electroplaxes being stained according to the direc- tions given in the first part of the paper. The results were the same as with the iron hematoxylin, namely, the less dense portions of the electroplaxes and the rods had disappeared. The fact therefore that these rods were disgested in 3 minutes by a weak solution of pepsin discredits absolutely the hypoth- esis that the rods are composed of elastic connective tissue, for elastic fibers are digested only very slowly by pepsin, the time required being several hours; the control in this case being the same as that noted above-the ligamentum nuchae of a horse. Having shown that the rods are not composed of connect- ive tissue of any sort, the next most logical hypothesis was that they were of some form of keratin, chitin, or chondrin, and the following test was performed accordingly. Vertical sections of the electroplaxes of Astroscopus, after the paraffin had been removed, were put for 24 hours in a 72 per cent solution of hydrochloric acid. The acid was then washed out and the sections stained with iron hematoxylin and then examined with the microscope. The rods could not be found, but hollow spaces corresponding to the shapes of the rods were found in the electric layer of the electroplax. Figure 3, a drawing of a slide after treatment with hydrochloric acid, shows these spaces clearly. The spaces are seen to be a little wider and larger than the rods, showing that the rods must have swelled to a certain extent before being dissolved by the acid. This latter statement was also confirmed by treating the rods with the acid for only a short time, and then examining with the microscope; the result being that the rods had swelled to a considerable amount. The other elements of the electro- plax, nuclei, etc., in their general form remained intact. There- ELECTROPLAX OF ASTROSCOPUS 105

Fig. 3 Oblique section through an electroplax treated with 73 per cent hydrochloric acid. The portion of the electric layer shows the electric nuclei, and also the spaces which were occupied by the rods before they were dissolved. The spaces have a greater diameter than that of the rods because the rods swelled before being dissolved. X 1200.

fore this indicates clearly that the rods are not composed of chitin or keratin, for these substances are not attacked by hydrochloric acid of the above strength. To strengthen this statement we have only to quote the results from the pepsin treatment noted above, in which the rods were digested in 3 minutes. They could not therefore have been composed of chitin or keratin, for according to Encycl. mikr. Technik, these substances are not attacked at all by pepsin. From the above facts therefore we may with a good degree of certainty con- clude that the rods are not composed of any kind of chitinous or keratinous substance. Having found that the rods do not consist of any kind of connective tissue or of chitin or keratin the only reasonable hypothesis left was that they were some forni of muscle fiber, not having however the same chemical com- 106 JAMES G. HUGHES, JR. position as the ordinary striated fiber, from which the electro- plax is derived. Accordingly the following stains were applied: (1) Van Gieson’s picro-nigrosine, which stains muscle a yellowish-green and connective tissue blue, mixed as follows : To 45 cc. of saturated aqueous solution of picric acid, 5 cc. of 1 per cent aqueous solution of nigrosine were added, the whole mixed thoroughly. Paraffin sections of the electroplaxes (ver- tical) fixed in pure corrosive sublimate were stained for 12 hours in this mixture; then wztshed in picric alcohol, dehydrated rapidly, cleared and mounted. A description of an electro- plax is as follows: The jelly electric tissue stained blue. The electroplax proper with its three layers stained a yellowish- green. The rods were just visible, and were stained the same color and to the same degree as the electroplax. The fact therefore that the rods stained the same coldr as the electro- plax and as muscle shows that they are very probably com- posed of some muscle-like substance. It may be noted here that the electroplax proper always stains about the same as voluntary muscle, a control consisting of voluntary muscle hav- ing proved the truth of this statement several times. The reason for this is apparent when wc recognize the fact that the electroplax is derived from a striated muscle fiber. This stain also confirms the results of Weigert’s resorcin-fuchsin stain that the rods are not composed of any kind of connective tissue, and also indicates decidedly that the jelly electric tissue is white fibrous connectJive tissue. The controls used were the adductor muscle from an oyster, the ligamentum riuchae of a horse, and the white fibrous connective tissue in the umbilical cord of a sheep, all being stained exactly the same period of time as the electroplax. (2) Van Gieson’s picro-fuchsin, which stains muscle yellow and connective tissue red, was mixed as follows; To a satu- rated solution of picric acid were added a few drops of a sat- urated aqueous solution of acid fuchsin, until the mixture became a deep red. The stain was then ready for use. (It may be noted that, if too much acid fuchsin be added, muscle as well as connective tissue will stain Ted). ELECTROPLAX OF ASTROSCOPCS 107

Paraffin sections of the electroplaxes (vertical) fixed in pure corrosive sublimate were stained for 6 hours in the above mix- ture. The results were as follows: The jelly connective tissue stained red, the electroplax proper with its three layers stained yellow. The rods were scarcely visible, staining the same color (yellow) as the electroplax. The fact that the rods stained yellow would indicate again that they are composed of muscle tissue, and this in connection with the picro-nigrosine stain in which the rods stained the same color as muscle gives us strong ground for believing that the rods are composed of muscle tissue; probably involuntary muscle fibers as the rods are not striated. The fact also that the jelly electric tissue stained red shows again that it must be of the same composition as ordinary white fibrous connective tissue. The controls were the same as those for Van Gieson’s picro-nigrosine. It may be objected that the rods did not stain the same color as the electroplax proper with Mallory’s connective tissue stain. To explain this we may say that the rods stained a brilliant red, which is the color that muscle tissue should take in the above stain. The fact that only the electroplax stained a reddish-purple shows that the rods are more strictly com- posed of a muscle substance than the electroplax itself. The only tissue now left of which the rods might be com- posed is nervous; that is, the rods might be nerve endings of some kind, which they resemble a little. In order to test this Paton’s silver nitrate stain for demonstrating nerve fibers and endings was applied and the results carefully noted. The stain and fixation were as follows: The electric organ was fixed in 10 per cent formalin solution, neutralized with magnesium carbonate. It was then cut in small strips about 4 mm. thick. These were washed with running tap water for 12 hours and then in three changes of distilled water for about 30 minutes. The tissue was then put in 1 per cent silver nitrate solution for 6 days in the dark. The tissue became a reddish-brown in color. (It may be noted here that if the tissue becomes a yellowish-brown the stain has not been applied correctly, and it is advisable to throw the whole speci-

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 1 1 08 JAMES G. HUGHES, JR. men away.) It was then placed in a freshly prepared solution of silver nitrate, made as follows: To 20 cc. of 1 per cent silver nitrate solution, two drops of a 40 per cent solution of caustic soda were added. A gray pre- cipitate was formed. Twenty to thirty drops of strong am- monia were then added-just enough to dissolve the precipitate. The tissue was allowed to remain in this for 45 minutes; then washed in distilled water containing a few drops of glacial acetic acid (25 drops of acid to 100 cc. of water). It was left in this for about 10 to 15 minutes, until the reddish-brown color had changed to yellowish-brown. It was then washed in distilled water, and placed for 12 hours in 1 per cent hydro- quinone containing 5 per cent neutral formal. It was again washed with distilled water, dehydrated, and imbedded in paraffin through chloroform. The tissue was then cut and sections mounted with balsam. A description of a vertical section of an electroplax so stained is as follows: The electroplax proper stained a very light brown. The nuclei, staining a little deeper, could be clearly seen. The electric connective tissue did not stain at all, and was not even visible. The nerve endings, which were very abundant, stained dark brown or black, thus being clearly differentiated from the rest of the electroplax. The rods could not be found. If present, they must have stained light brown, the same color as the electroplax, for they certainly were not present as nerve endings, which as I noted above, stained a dark brown or black. This conclusion therefore is evident, that the rods are not nerve endings of any kind. The fact therefore that these fibers and rods have been shown to be composed of a muscle-like substance brings up a new kind of muscle fiber to the attention of histologists. These fibers have not been shown to be contractile, are non-striated, dense and exist without any apparent connection with each other. In view of the above facts we cannot give to these ele- ments of the electroplax any other function except that of sup- port, and this will consequently have to suffice until some other investigator presents a better interpretation.