Talk:Book - Contributions to Embryology Carnegie Institution No.35

CONTRIBUTIONS TO EMBRYOLOGY, No. 35.

MUSCULAR CONTRACTION IN TISSUE-CULTURES.

By Margaret Reed Lewis, Collaborator in the Deparlmenl of Embryology, Carnegie Institution of Washington.

With two plates and six text figures.

CONTENTS.

PAGE.

Method 193

Types of muscle 194

Smooth muscle from the amnion 194

Growth from the amnion in tissue-cultures 194

Contraction of the smooth-muscle cells 197

Comparison of the growth in tissue-cultures with cells of the normal

amnion 199

Fixed preparations 200

Heart muscle 201

Growth from the heart muscle in tissue-cultures 202

Contraction of the heart-muscle cell 202

Comparison with cells of the normal heart 203

Skeletal muscle 203

Growth from the skeletal muscle in tissue-cultures 204

Contraction of the skeletal-muscle cell 205

Other cross-striated muscular tissue 205

Discussion 207

Conclasion 209

Bibliography 210

Description of plates 212

192

MUSCULAR CONTRACTION IN TISSUE-CULTURES.

By Margarkt Reed Lewis.

The solution of the problem of the structure and behavior of muscular tissue has been frequently undertaken either from the standpoint of detailed and com- plicated cell architecture or from that of the mechanics of dead material. It is by no means claimed that the observations herein recorded solve this most interesting problem; nevertheless the results show that it is possible to consider the question entirely from the standpoint of hving material and at the same time to be able to disregard to a great extent the comphcated structure of differentiated muscular tissue. In other words the mechanism to be observed can be reduced to the simple terms of a single sUghtly differentiated, U\dng cell maintained under observation throughout experimentation.

METHOD.

Although a new procedure, the tissue-culture method is well known, in view of the fact that when it was first used conclusive results were obtained in two of the important problems of anatomy and physiology— t. e., that the axons grow out from the nerve cells (Harrison, 1910) and that the heart muscle is capable of inde- pendent contraction (Burrows, 1912). The method used in the present experi- ments, while similar in most respects to that of Dr. Harrison and also that of Dr. Burrows, was devised for the study of the cells of the blood, bone marrow, and spleen, in relation to tuberculosis, independent of the work of the above authors. The original solution contained agar. Since it was found (Lewis and Lewis, 1912) that the presence of agar is not necessar}' for growth of the cells, the Locke-Lewis solution, modified (M. R. Lewis, 1916) according to the species of animal and also according to the osmotic pressure of the tissue to be explanted, has been employed.

Tissues from chick embryos of 4 to 12 days' incubation were used for the cultures. The medium was Locke-Lewis solution (90 c.c. of NaCl 0.9 per cent + KCl 0.042 per cent + CaCL, 0.025 per cent + NaHCOj 0.02 per cent -|- 10 c.c. of chicken bouillon -f 0.25 per cent dextrose; Lewis and Lewis, 1915). Aseptic conditions were maintained throughout. The embryo was removed from the egg and placed in a petri dish containing 20 c.c. of warm solution. Pieces of the tissues to be explanted were removed, washed through one or more changes of warm medium, and cut up with sharp scissors into pieces about 0.5 mm. in diameter. Each piece was then placed on the center of a cover sUp, part of the drop drawn off, and the cover slip sealed on to a vaseUne ring around the well of a hollow- ground slide. Cultures thus prepared were kept in an incubator at 39° C. All microscopical observations and experiments were made in a warm box at 39° C.

The cells began to migrate out from the explanted piece within a few hours. At the end of 18 hours a zone of cells, usually only a few cells deep but often wider

193

194 MUSCULAR CONTRACTION IN TISSUlE-CULTURES.

than the cx[)hinted piece, was formed. Among the cells of this new growth were man,v mitotic figures (figs. 7, 8, and 9). As the cells migrated out they became spread out more and more closely ujjon the under surface of the cover slip, so that the edge of the growth was composed of a single layer of large, flat cells. These flat cells, although somewhat distorted in so far as the position of certain cyto- plasmic structures are concerned, have an advantage over those obtained bj' means of sections, in that all the structures of the cell are present and can be observed in their relations throughout the activity of the Uving cell. The region between these outer cells and the explanted piece may be one or several cells in depth. The cells here, while largely spread out, resemble more nearly the cells of the normal embryo and are very httle distorted laterally.

8i)ontaneously contracting cells usually were found among the less spread- out cells and not among those at the edge of the growth. Cultures of the amnion furnished numerous rhythmically contracting smooth-muscle cells ; those of the heart gave rise frequently to sheets of cells contracting in co-ordination with the beat of the explanted jiiece, and at times to a few isolated cells beating independently; while from the skeletal muscle were obtained muscle buds, muscle-fibers, and myoblasts, each of which were occasionally found undergoing spontaneous contraction.

For a better understanding of the cells in tissue-culture various preparations of other li\dng muscular tissues were made, among which may be mentioned the following: (1) The entire uninjured amnion of a 3, 4 or 5 day chick embryo; (2) a 2 to 3 daj^ chick embryo with beating heart ; (3) preparations of teased heart muscle and teased skeletal muscle-fibers of chick embryos; (4) certain microscopical marine copepods whose cross-striated muscle-fibers could be studied while the animal remained alive; (5) the isolated sarcostyles of the insect's wing muscle; (6) thin slices of the muscle-fibers from an adult dog, cat, or turtle. In addition to the above, preparations were fixed and stained in various ways in order to compare the results with those of other investigators.

TYPES OF MUSCLE. SMOOTH MUSCLE FROM THE AMNION.

GnOWTH FROM THE AMNION IN TiSSUE-CuLTURES.

The amnion, as is well known, consists of a layer of smooth-muscle cells over- lying a layer of epithelial cells, in neither of which have nerve-cells or fibers been satisfactorily demonstrated (fig. 1). The outgrowth from this tissue in cultures did not usually form a membrane composed of the two ty]x\s of cells, but instead the smooth-muscle cells (sm) and the epithelial cell (e) grew out more or less independently of each other (fig. 9). No nerve-fibers were found in any cultures from the amnion, regardless of the age of the chick. This behavior is quite contrary to that of cultures from certain other muscles (heart, stomach, and intestine) in that a large jiercentage of cultures of the heart and intestine contain nerve-fibers. The lack of growth of nerve-fibers may be taken as an indication that no gangUon cells resembhng those of the heart or intestines were present in the

MUSCULAR CONTRACTION IN TISSUE-CULTURES.

195

explanted pieces of amnion. The epithelial cells were readily distingui.shable from the smooth-muscle cells, since they became spread out as large, flat, more or less hexagonal cells, frequently united together in the form of a membrane (e), while the muscle cells were either in the form of slender bands or large flat cells (sm) decidedly elongated in the direction along which migration had taken place (fig. 9). In either case the muscle cells were characterized by a pecuUar refraction of the cytoplasm due to some substance which took part in its constitution. This phenomenon is exhibited to a certain extent by all the cells in tissue cultures— for instance, where a process of a cell is curled in active movement. All types of muscle cells, however, have a much greater refraction than have other cells. This characteristic increased coincidentally mth the maturing of the cell and was especially marked in cells which were undergoing rhythmical contraction. Levi (19166) mentions that the muscle cells from the heart can be distinguished from the mesenchyme cells by a difference in their opacity.

Fig. 1. — Normal amnion from a 6-day chick embryo. Film preparation. Silver nitrate. Heavy lines indicate smooth-muscle cells. Dotted lines show the epithelial cells.

The elongated, band-like, smooth-muscle cells were usually found near the explanted piece; they practically always exliibited rhythmical contraction. The large, flat cells were located mostly along the edge of the growi^h and seldom con- tracted rhythmically unless stimulated to do so. The shape and behavior of a cell was determined to a large extent by its position in the growth. A band-Uke cell one day undergoing rhythmical contraction might the next day, through migration, have become one of the large, thin cells along the edge of the growth.

Many of the large, flat, smooth-muscle cells displayed a marked tension along the cover-slip, so much so that, in some instances, the cytoplasm was drawn into slight folds which frequently extended from one cell to another, through the long axis of adjacent cells, as marked intercellular bridges. Many neighboring cells were joined also by more dehcate lateral processes. This hving growth, however, is not a syncytium in the usual sense of the term, for even the most pro- nounced connections between the cells were frequently withdrawn during the migration of a cell away from its neighbors, or by reason of the mitotic division of the cell. It was possible to cause the withdrawal of the intercellular bridges by various experimental procedures; for instance, a minute amount of glycerine intro- duced into the neighborhood of the growth caused all cell processes to be immedi- ately withdrawn so that the cells became isolated individual cells and remained so

196 MUSCULAR CONTRACTION IN TISSUE-CULTURES.

for several hours after the abiiornial eiivironincnt had l)e('n removed. This reaction of the cells may account for tiie failure of certain observers to find coimections between the smooth-muscle cells. Certainly the methods of teasiiiff the pn^para- tion, described by Hchaffer (1899), may account for the fact that isolated or i)artly teased-out cells in his preparations did not possess intercellular bridges.

No myofibrils were observed in the cells of the living cultures. Except where lines of tension were evident, the cytoplasm appeared as a homogeneous substance in which were embedded granules of different sorts. Very sHght disturbances of the cell, however, led to the formation of threads within the cytoplasm. All fixed muscle cells contained fibrils of varying thicknesses located over the surface, where no threads could be distinguished previous to fixation. The appearance of these Unes corresponds exactly to what has been described by other observers (Verzar, McGill, Benda, etc.) as myofibrils, although no myofibrils were present in the living cells. Thus the living smooth muscle is characterized by some sub- stance within the cytoplasm which possesses a pecuhar refraction, while the dead tissue contjiins the typical myofibrils. In all probability the myofibrils exist in the living cell in the form of this characteristic material.

That the fibrils are formed by the coagulation of the cytoplasm can be observed directly by watching under the microscope the influence of various substances upon the Kving cell. For instance, the progressive action of a minute drop of dilute lactic acid placed on the border of the culture drop of medium caused the formation of lines of coagulation, accompanied verj^ shortly b.v the death of the cell. This occurred first in the outermost cells and later in the thicker cells near the explanted piece. While both the coarse and the fine fibrils appeared in most of the cells upon fixation, the fine fibrils predominated in the more spread-out cells and the coarse fibrils were more evident in the thicker cells. Figure 17 is a careful drawing of a cell after the formation of the fibrils. Before fixation there were no fibrils present in the cytoplasm. The only indications of tension were slightly more refractive regions at the two ends of the elongated cells and a dim line across the nucleus, as though the cytoplasm had been drawn slightly tliicker in the long axis. The mitochondria were clearly distinguishable as shiny, slightly wavy filaments. These bright threads did not remain in any one shape but became more or less wavy, occasionally .sei)arated into shorter lengths or united together again, and, in short, exhibited activity characteristic of mitochondria in the cells of tissue cultures. This activity demonstrates that there was no structure i)resent in the protf)i)lasm sufliciently dense in nature to interfere with their movement. Upon fixation the protoplasm formed numerous coarse and fine threads, while the mito- chondria did not undergo any change. The threads of coagulated protoplasm are quite different in ajipearance from the mitochondria (fig. 17). The coarse fil)rils spread out into finer ones in much the same manner as that depicted by McGill (1907, fig. 23). Tliis appearance is such as to suggest that the fibril was due to coagulation.

Those cells in process of mitosis before explantation completed their division in the cultures. Other cells along the edge of the pieces divided, so that, from the

MUSCULAR CONTRACTION IN TISSUE-CULTURES. 197

beginning of growth, mitotic figures were found here and there among the migrating cells. While mitosis of the contracting cells was observed in only one case, it wa.s a frequent occurrence among the large flat cells. The process took place in the same manner as has been described for various cells in tissue cultures (Lewis and Lewis, 1917f). During the division of the cell, fibrils -were not formed across the cell upon fixation. Just what happens to change the behavior of the contractile tissue at this time is not known, but it is probably involved with the factors at work in the phenomenon of division. The daughter-cells, however, contain fibrils after fixation. Champy (1914), through observations upon fixed cultures alone, found that the dividing cells did not contain myofibrils. From this he advanced the theory that differentiated structures become lost in the growth in cultures due to the rapid mitosis of the cell. This is certainly not true in the cultures of the amnion, for the cells retain their ability not only to contract, but also to form fibrils upon fixation in spite of the fact that mitosis frequently occurs. Epithehal cells in the same cultures did not exhibit the same refraction as did the smooth-muscJe cells, nor were fibrils formed in them upon fixation, even when an epithelial cell wa.s .side by side with a smooth-muscle cell (fig. 12).

CoNTHACnON OF THE SmOOTH-MuSCLE CeLLS.

Various observers have shown that it is possible for smooth-muscle cells to undergo rhythmical contraction when isolated from the body, either as rings or as strips taken from organs containing smooth muscle (Stiles, 1901; ]\Iagnus, 1904; Langley and Magnus, 1905; Roth, 1907; McGill, 1909, etc.). In the experiments of the above authors it was found that various agents stimulated while others inhibited the phenomenon. For general observation, however, Locke's solution was found to be the most favorable medium. The experiments in tissue cultures, wl^le demonstrating unquestionably that it is possible for smooth muscle to undergo rh^'thmical contraction when entirely separated from the nervous

system, have in addition the value of Fig. 2.— a bundle of three contracting cells. The contrac-

permitting the observer to follow the *'°° "'^'^ '^ indicate by the dotted line. Culture 24 hours

' ^ old from amnion of 5-aay cluck embrj-o. Oc. 6, lens 3 mm.

phenomenon under the microscope

and to see just what changes take place in the structure of the indi\idual cell.

The process of contraction of the anmion cells in tissue cultures may be exhib- ited by several cells, by a single cell, or by only a portion of a cell. There seemed to be present in the cell some active change which caused the protoplasm to be drawn towards a given region. In everj^ case there was a quiet region bej-ond which no further movement of the protoplasm took place. The result of tliis current of protoplasm was that the cell became swollen in the region of active change, and usually this area was thrown into folds. The phenomenon (current of protoplasm drawn towards a given region) was exhibited in the same manner many times. The region to which the protoplasm was drawn, and usually piled in

198 MUSCULAR CONTRACTION IN TISSUE-CULTURES.

folds, was a more or less definite one; /.<., roughly .speakinfj;, the process was repeated in the same region at a given rate per minute for longer or shorter intervals of time (fig. 2) . The area of contraction corresponds with the contraction node described by AlcCJill, as will be shown later. It was not, however, merely an accidental i)oint at which a contraction wave passing through the cell happened to be fi.xed. The active change then appeared to be neutrahzed and the protoplasm returned to its former position only to be again drawn towards the same spot after a more or less definite interval of time. In figure 13 a contraction area was located at C in each of three different cells and a rhythmical contraction took place there at a rate of 8 per minute. That the jirotoplasm actually fiowed was demonstrated by the movement (in addition to that of the contractile material) of the nucleus and granules in some cases, and of the granules only in others. The cytoplasm was piled in folds at each C and did not appear as shown in the drawing until after fixation. The contraction node was an expression of the contraction of the cell. There was no wave of an enlarged area sweei)ing over the cell as is present in the intestine during peristalsis, neither did the behavior appear to be due to a narrower ring passing along the cell. The activity could be described only as that caused by the attraction of protoplasm to a given region.

A number of observers— Remak (1843), Ley dig (1849), Schwalbe (1868), Rouget (1881), Marshall (1887), Werner (1894), Schultz (1895)— have described folds in the smooth-muscle cells. These writers claimed that the smooth muscle shortened by a series of zigzag foldings. Such a process does not agree with that displayed by the amnion cells, for in the latter the jirotoplasm was moved from one region of the cell to another. The cell became larger and was thrown into folds in the region to which the protoplasm was drawn, accomj^anied by a decrease in the size of the cell in the regions from which the protoplasm was taken. A zigzag shortening might appear to have taken place where the entire muscle cell was drawn into the contracted area, as in the contraction of the total amnion noted below, so that the protoplasm of the whole cell was contained witliin the enlarged and folded area. This area might then appear as though shortened by means of a folding in of the protoj)lasm. KoUiker (1849), Ileidenhain (18()1), SchalTer (1899), Grutzner (1904), and SoU (1906) described knob-hke thickenings along the surface of the unfixed muscle where the protoplasm, although not folded, is thicker than the remainder of the cell. Such behavior corresponds more nearly with that produced by exposing the .smooth-muscle cells in cultures to some abnormal environ- ment rather than to the folded area of normal contraction. In the former case knob-like .sw(>llings appear along the surface of the cell and remain for .some time.

No change, other than a slight thickening and shortening, was exhibited by cither the nucleus or the granules relative to each contraction. Fre(}uently pseudopodia were extended out from the cell or withdrawn into the cell during that period of time in which rhythmical contraction was exhibited. Accompanying the flowing of the cytoplasm toward the region of active change was a sway- ing or pendular motion which twisted the cell about the long axis. The twisted and bent nuclei described by Van Ciehuchten (1889), Ileidenhain (1900),

MUSCULAR CONTRACTION IN TISSUE-CULTURES. 199

Forster (1904), and McGill (1909) may be due partly to this. In one case, where several cells formed one long strand wliich wa.s undergoing rhythmical contractions, the pendular movement was so violent that it terminated by one of the cells near the middle of the strand being whirled completely around several times, much as a spool upon two twisted strings whirls about when the latter are loosened and then again pulled taut (fig. 3).

Comparison- of Tissue-Culture Growth with Celi.s of the Normal .\MNno.v.

Preparations for a study of an uninjured amnion can be made as follows: The entire blastoderm of a chick (72 to 96 hours incubation) is stretched out on a cover- slip moistened with Locke-Le^Nds solution. This is then inverted over a hollow ground slide and sealed with vaseline. Another successful method is to place an older embryo in a deeper well and impose the cover sUp directlj^ upon the extended amnion. In either case it is important to permit an air space to remain around the embrj'o, otherwise the contractions will shortly cease. The cessation of activity' of the amnion which results when the preparation is entirelj'^ covered \nth medium may, in some measure, be due to the action of carbon dioxide, since Hooker (1912) finds that oxygen is essential for the rhythmioity in vascular muscle. According to Dr. Hooker, if the muscle is exhibiting rhythmicity, this is abolished or depressed by CO2.

Contraction and relaxation of the smooth muscles of the normal amnion resulted in a rocking or swaying motion. This is due to the presence, in various regions throughout the amnion, of a peculiar, star-shaped arrangement of the muscle fibers (fig. 15). Fiilleborn (1895) gave a short description of the muscle fibers of the amnion. This author states that the muscles of the amnion of a chick of 5 to 6 days' incubation are short, spindle-shaped cells. During the first half of the development of the embryo these cells grow into long, slender bands. In certain regions these cells are arranged into large and small, star-shaped groups from which the muscles stream out in all directions. Verzar (1907) later pubhshed a description of this same star-shaped arrangement, together with an analysis of these muscle centers and their relation to the motion characteristic of the amnion. In the growth from the amnion in tissue cultures this peculiar configuration of muscle fibers was found only among the cells in the immediate vicinity of the explanted piece.

=^^5>-

Fig. 3. — A living smooth-muscle cell which was whirled .about the muscle strand as the result of the pendular move- ment. Culture 48 hours old, from amnion of S-daj- chick. Oc. 6, lens 3 mm.

Contraction of the normal amnion did not usually involve the entire amnion at any one time. Such activity was exhibited by the cells throughout quite an extensive region. When these cells relaxed the phenomenon was repeated in the same or another area. The cells were drawn together mth a swajdng motion, so

200 MUSCULAR CONTRACTION IN TISSUE-CULTURES.

that the cells, thrown into folds, heeame much shorter and thicker than the}' were previously. At times when the action of the amnion was weak, onl}' a small group of cells, or sometimes merely a single cell, underwent rhythmical contraction. In such cases the behavior of the active cells was identical with that exhibited by the cells in tissue cultures. The contraction of the normal amnion, even when very extensive, was usually characterized by a cjuiet region beyond which no further movement occurred, together with the folds of muscle protoplasm in the area of the active center. This motion was always accompanied by a slight swaying or pen- dular motion. In other words, while the active region of contraction of the normal amnion was usually occupied by many cells, the phenomenon of contraction, nevertheless, corresponded with that of the cells of tissue cultures.

Fixed Preparations.

When cultures of amnion cells were fixed they became entirely changed in appearance, and they then exhibited a striking resemblance to the results given by other observers for the normal chick amnion. In many of the cells the coagula- tion took place in such a manner as to dupUcate the structure shown in figure 4 by \'erzar. In these cells it was diflRcult to distinguish the mitochondria from the myofibrils, owing to the manner in which the two structures were intermixed, so that it was not surprising that Verzar classed both bodies as myofibrils. However, in cells a Uttle more spread out laterally (fig. 16) it became a simple matter to dis- tinguish between a thread due to the coagulation of the cytoplasm (myofibrils) and the mitochondria, because of the fact that the mitochondria w^ere usually wavy, not straight, the same width throughout, not varying, and ended abruptly instead of branching out into finer threads, as did the fibrils. The centrosomc is ([uite clear in the fixed, spread-out cell, and corresjionds with that described by Lenhossek (1899), in that it Ues near the nucleus and appears to be double or dumb-bell shaped.

^^■llile it must not be forgotten that in these cultures of smooth muscle only embryonic amnion tissue is dealt wdth, nevertheless, it seems as though a few of the results may be compared with those of adult tissue in such a way as to lead to a better understanding of smooth-muscle tissue in general. For instance, figures like many of those which Miss McClill has shown for other types of smooth muscle can be produced in the culture of the amnion by using the jjroper method of fixa- tion. A comparison of the coarse and fine fibrils obtained by other investigators with the aj)i)earance produced in the smooth-muscle cell by the coagulation of the cytoplasm shows that in all probability the structures are the same. Take, for instance, where Miss ]\Ic(;ill (1907(;) states from her figure 23 that the coarse fibers are bundles of finer fibrils; a study of these amnion cells leads to the conclusion that the process by which this appearance was formed may have been the same as that by which the coarse and fine fibrils of figure 16 and 17 were formed. In other words, the fibrils did not exist as such in the living cells, but some material capable of producing such structures upon coagulation was present.

A number of instances have occurred where the substance along the fine of tension extending between cells was coagulated in such a manner as to give the

MUSCULAR CONTRACTION IN TISSUE-CULTURES. 201

appearance of coarse fibrils passing from cell to cell, as described bj- Benda (1902) and McGill (1907a, 1909). An interesting picture was obtained by fixing a culture with 10 per cent nitric acid and later staining it (fig. 14). In this case the edge of the cells became coagulated in such a manner as to imitate the appearance described by McGill (1909, fig. 15) as "heavy, elastic fibers" and "the more delicate con- nective tissue network." However, in this case the dehcate connective-tissue network was formed by coagulation of traces of the rapidly withdrawing inter- cellular bridges and the elastic fibers by the fixation of the curled edges of cells just beginning to retract. Such behavior is not without precedent in the fixation of Hving material. The work of Levi (1916a), Lewis and Robertson (1916), Lewis and Lewis (1917c), and Chambers (1917), etc., has demonstrated that the actual threads of the mitotic spindle are not present in the living cell. Cowdry (1914) states that the Nissl substance does not exist in the Uving nerve cell. Marinesco (1912), Mott (1912), and Lewis and Lewis (19126) claim that neurofibrils are caused by the fixation of the nerve fiber. This by no means infers that myofibrils are artefacts ; it merely claims that the substance formed bj' the cell during differentia- tion does not necessarily exist as threads in the Uving cell, but that it assumes this form upon the coagulation of the cytoplasm. At least it can be demonstrated in these smooth-muscle cells of the amnion that threads (myofibrils) appeared upon the fixation of the living cell, where none existed previously.

So far it has proved impossible to fix the area of contraction as it appeared in the living cell, even in cases where the fixing solution was injected through an opening in the ring of vaseline directlj^ upon the contracted cell. As soon as the fixative touched the cell the folds disappeared, although the protoplasm remained thicker and more concentrated in this region. Coincident with tliis, more or less straight, coarse fibrils were formed (fig. 13). After fixation the appearance of many of the cells was such that it might easily lead to an erroneous conception of the phenomenon of contraction; namely, that it had been caused by a shortening and thickening of the myofibrils in such a manner as to concentrate the protoplasm in this region. When fixed contracted muscle cells were stained (fig. 13), the area of contraction, in which folds had been present before fixation, appeared much the same as that structure termed by McGill (1909) the "contraction node." Con- cerning this, Miss McGill states :

"During contraction more changes take place in smooth muscle than can be attributed to morphological causes, such as thickening of the myofibrils, etc. At the contraction nodes the staining reaction would indicate that there is a marked chemical reaction taking place also."

This view coincides with that expressed above, that there is a center of active change.

HEART-MUSCLE.

Burrows (1912) first described the gro^^'th and contraction of the muscular cells arising from pieces of embryonic chick heart explanted in plasma. According to Dr. Burrows, these cells do not contain cross striations. although they undergo rhythmical contraction for 24 to 96 hours. In the new growth there could be

202 MUSCULAR CONTRACTION IN TISSUE-CULTURES.

distinguished certain cells that were sei)arated from their neighbors. Each one of these isolated cells contracted with a rhythm quite different from that of the rest of the e.xplanted piece and, moreover, the rate was not necessarily the same in each cell. Lake (1916) described the independent rhythmical contraction of the cells from the heart in j^lasma, but the results of his observations have added notliing to the facts previously pubHshed by Dr. Burrows. Shipley (1916) found that in cultures of the anlage of the chick heart "the embryonic cell which is des- tined to become heart muscle will differentiate and begin to function even, though removed from its normal environment."

Prior to the experiments of the above investigators, demonstrating the actual contraction of the single heart cell, Gaskell (1882) had shown that in all probability the heart muscle is capable of beating without stimulation from the nervous system. This observer claimed that not only does the beat arise spontaneously in muscular cells, but also that the conduction of the excitation from one part to another takes place through muscular tissue. The action of certain salts upon isolated muscular tissue from the heart has been discussed by Howell (1898), Lingle (1900), and others. The salts shown by these investigators to be necessary for the rhythmical contraction of this tissue are present in the Locke-Lewis solution.

Growth from the Heart-Muscle in Tissue-Cultures.

The growth arising in Lock-Lewis solution from explanted pieces of chick heart (4 to 6 days' incubation) differed only slightly from that described b}^ Bur- rows (1912). It tended to form a membrane wliich beat as a whole, so that isolated contracting cells were less frequently seen (fig. 7). The cells were joined together by cytoj)lasmic bridges extending from all sides. These bridges were not perma- nent but were formed or withdrawn coincidentl.y with the movement of the cells (figs. 12, 13, and 14, Lewis and Lewis, 1912a). During mitosis the cell rounded up and remained attached to its neighbors by only a few deUcate, hair-Uke processes. The plane of division separated the cell into two daughter-cells. Schochaert (1909) found that although the embryonic heart muscle appears to be a syncj'tium, in reality it is composed of jirimarily individual cells, since during mitosis the spindle plate is formed, indicating that the heart-muscle cell divides into two cells. There was no evidence of cross-striation in the living cells. Occasionally, upon fixation of the cultures, the cytoplasm became coagulated into fine Unes over the surface of the cells. In the few fixed heart-muscle cells studied no tj^pical cross- striations were observed. Levi (19166), however, describes the development of cross-striated fibrils in the growth from the heart in plasma cultures.

Contraction of the Heart-Muscle Cell.

The beating cells were smaller than those of the amnion and remained more nearly oval in shape instead of becoming str(>tched out into sl(>nder bands. Each of the cells observed during contraction cxliibited activity throughout the entire cell, never in one portion only. The cytoplasm was drawn towards the center of the cells without being thrown into folds, and resulted in a bellying out of the cell as a whole. This was accompanied b^^ a slight pendular movement. The rate of

MUSCULAR CONTRACTION IN TISSUE-CULTURES.

203

the rhythmical contractions was rapid, about 70 to 120 per minute. Owing to the rapid shortening and thickening, together with the slight pendular movement, the phenomenon of contraction exhibited by the few entirely isolated cells observed had the character of a distinct beat as though the single cell constituted in itself a minute force pump rather than an infinitesimal part in such a structure. Figure 4 shows a few of the changes of form through which an isolated contracting cell passed within the period of a few hours. Tliis cell, although exhibiting contrac- tions at the rate of 115 per minute, formed several pseudopocha while under observa-

FiG. 4. — Changes in shape exhibited by a heart-muscle cell while undergoing rhythmical con- traction. Drawn at intervals of 1.5 minutes. Rate 115 beats per minute. Culture 48 hours old from heart of 4-day chick em- bryo. Oc. 4, oil- inim.

tion. The cell was markedly refractive, there were no myofibrils present, and the mitochondria and other granules were neither markedly different from those of any other cell, nor did they undergo any change relative to each contraction of the ceU.

COMP.\RISON WITH THE CeLLS OF THE NoRM.\L HeaRT.

An effort was made to compare the behavior of the heart cells in tissue cultures with those of the normal heart. Preparations of the entire blastoderm (2 to 4 days' incubation) were made in the same manner as that used by Sabin (1917) for the study of the development of the blood-vessels in the h^^ng chick embryo. The beating heart was observed with ease, but it proved to be practically impossible to analyze the part played by the individual cell because the coordination of the mass of cells was perfect. The coordination of the beat of the cells of the explanted piece, however, can be disturbed by the addition of calcium to the culture medium. In one such experiment each cell acquired an independent contraction, so that the result was an astonishing dancing of the individual cells without any coordinate beat of the piece as a whole. This pecuUar activity was caused by an extremely rapid shortening and thickening, together with a slight pendular movement of each cell.

SKELETAL MUSCLE.

Loeb (1899), Garrey (1905), Langley (1908), and Mines (1908) have each discussed the contraction exhibited by isolated skeletal muscle in various media. While the phenomenon continued for only a very brief interval of time in any of the media used by these observers, nevertheless Locke's solution was found to be the most favorable medium for experimental purposes.

204 MUSCULAR CONTRACTION IN TISSUE-CULTURES.

The question as to whether cross-striated muscle fiber can undergo regenera- tion in tissue cultures has been discussed by only a few observers. Sundwall (1912) found almost no proliferation of cross-striated muscle fibers from post partum animals. He states that at the end of 48 hours the cross-striations began to dis- appear and the terminations of the fibers became more or less globular in form. Even cultures of muscle tissue from 2 cm. embryos showed no growth resembling the original muscle fiber. Congdon (1915) observed that cultures from the limb bud of a 7-day chick embryo show the proUferation of a premuscle cell. The author (1915) gave a short description of the growth in tissue culture of skeletal muscle- fibers which exhibited rhythmical contraction. Levi (1916^) described the growth of the heart-muscle tissue and the pressence of cross-striated myofibrils in the cells of the new growth. In a few words he states that the growth from skeletal muscle corresponds largel}^ to that from the heart. Lewis and Lewis (19176) gave a detailed account of the structure and behavior of the cross-striated muscle in tissue cultures. These observers obtained the growth of new muscle fibers from the cut ends of the old fibers and, in addition to this, the development of many myoblasts. Certain of the regenerated fibers contained traces of cross-striation. Since then I have obtained as many as 20 to 30 regenerated muscle-fibers in a culture of skeletal muscle from 10-day chick embryos. These new muscle fibers extended out as far as twice, in some cases three times, the width of the explanted piece. Each of the regenerated fibers was cross-striated (fig. 11).

The fact that cross-striations are developed in the muscle cells of tissue- cultures must have an important bearing upon the question as to the nature of the growth in tissue culture. Champy and others contend that the cells "dedifferen- tiate" into an indifferent type, but if cross-striations can develop it appears as though, under proper conditions, the growth behaves as in regeneration.

Growth from the Skeletal Muscle in Tissue-Cultures.

The usual growth from an explanted piece of skeletal muscle from a chick embryo (8 to 10 days' incubation) consisted of connective-tissue cells, among which extended numerous muscle-buds, a few isolated muscle-fibers, and many scattered myoblasts (fig. 8). The muscle buds furnish an example of a true syncytium, as the protoplasm of the cells which form these structures is continuous. In fact, they have every appearance of being multinucleated cells. However, when such a muscle sprout was kept under observation it was occasionally found that a cell separated from the multinucleated mass and migrated away as an individual cell, thus demonstrating the probable aggregate nature of the muscle fiber. In addition to this, it has been shown that the protoj^lasmic ends of two separate muscle-fil)crs may sometimes fuse together (figs. 3, 4, 8, and 14, Lewis and Lewis, 1917/>).

The end of the muscle-bud is usually a large protoi)lasmic syncytium sjjread out along the cover slip, but the behavior of the muscle fiber (and also of the isolated mj^obla.st) is quite different from that dis[)layed by those from either the amnion or the heart, in that they seldom become spread out laterally into thin

MUSCULAR CONTRACTION IN TIS.SUE-CULTURES. 205

cells, even along the edge of the growth. Fixation causes the coagulation of the cytoplasm along the muscle fiber in the form of a more or less straight fibril. In the ends of the fixed muscle sprouts, however, many fibrils, both coarse and fine, are formed by the coagulation of this material (fig. 10) . These fibrils may be straight or curved in various ways, due probably to the amount of contraction of the mus- cular substance. In some musck; buds the coagulated material resembled the structure termed primitive myofibril by Godlewski (1902).

Contraction ok the Skelktai^Muscle Cell.

The skeletal-muscle tissue, whether in the form of a muscle sprout, an isolated fiber, or a myoblast, exhibited contraction as a rather rapid (3 to 120 times per minute, Lewis, 1915) shortening and thickening of the muscular material, with a tendency of the two ends to approximate each other. In the muscle fiber no circular folds were observed along the length of the fiber, neither was there any marked bellying out of the muscular protoplasm at any given region. No folds were found around the myoblasts, but there was a thickening along the middle of the cell (fig. 5). In no case was a pendular movement observed, either by itself or coincident with the shortening and thickening of the muscle cell. It might be stated that the phenome- non of contraction, as shown by the skeletal muscle, differed from that characteristic of the amnion cell and also from that exhibited by the heart cells, in that it was neither a flowing

, . , u i' /'I. i.\ ^" ^' — '"Skeletal myoblasts which were undergoing rythmical

(ammon) nor a beating (heart) contractions. Onecell witha rateof 4 per min.,the other

movement, but one that more nearly ^'^^ f '•^*«' "1 ^ p*^"" """• Culture .5 daj-s oW from skeletal

. "^ muscles of a 6-day chick embryo. Oc. 4, oil-imm.

resembled a straight twitch. Spon- taneously contracting muscle fibers, and also myoblasts, were frequently found, and in these the rhythmical contractions were exhibited for several hours. At times, how- ever, the activity was induced by some form of stimulation (M. R. Lewis, 1915).

Other Cross-Striated Muscular Tissue.

A number of the theories advanced to explain the phenomenon of contraction of muscular tissue have been based largely upon the reactions and appearances of the isolated sarcostyles from the wing muscle of the insect. The isolated sarcostyle proved a fascinating field for experimental investigation, as may be inferred from the numerous observations found in the writings of Krause (1873), IMerkel (1872, 1873, 1881), McDougall (1897), and Schiifer (1891, 1912); but it is difficult to understand why certain agents, such as acetic acid following alcohol (Merkel), should have been chosen to obtain results upon which conclusions were to be drawn concerning the action of living tissue. In my observations marked changes were brought about in the appearance of the sarcostyles by almost any change in the medium surrounding them—?', e., dilution, increase in osmotic pressure, increase in the amount of any one of the salts constituting the medium, or the addition of

206 MUSCULAR CONTRACTION IN TISSUE-CULTURES.

minute quantities of certain substances, such as acid, alkali, chloroform, nucleic acid, pancreatin, or magnesium sulphate (fig. 6).

Although the addition of weak acid, etc., to the medium did cause the side wall to bulge out, such swelling was not necessarily accompanied by a coincident shortening of the height of the individual sarcomere. Neither was the shortening and tliickening of the entire sarcostyle alwaj^s accompanied by a protrusion of the wall of the sarcomeres. Either the membrane of Krause or the Une of Hensen may become extended to a marked degree. Various degrees of extension of the Krause membrane were shown in the same sarcostyle. When the sarcostyle was placed in certain solutions all cross markings disappeared. The details of these experiments will be pul)lished in a separate paper, since they throw no light upon the jihenome- non of contraction exliibited by muscular tissue in cultures.

Exceedingly thin pieces of the skeletal muscle of the cat, dog, chicken, or turtle, cut with sharp scissors along a Une parallel to the length of the fibers, were mounted in a drop of warm Locke-Lewis solution. When such preparations were examined

Fig. 6. — Somewhat diagrammatic

representations of a few of the

many interesting shapes exhibited

by the isolated sarcostyles of the

— ^ house fly. A, normal sarcostyle

"""T"'""""- in fly plasma. B. swollen sarco-

™,«^ — meres in slightly acid Locke's

-..,™!!Zr"'"""' solution. C, isolated membranes

__;'■"-.««„ of Krause floating in Locke's

""""" — """"' solution saturated with magnes-

......„_^^] ium sulphate. D, fibrillatedsarco-

— -"" meres in Locke's solution con-

E taining nucleic acid. E, extended

lines of Hensen in diluted white of

egg, to which a minute crystal of

sodium chloride was added.

under the microscope in the warm box, contraction waves were found occurring in the individual fibers. The contraction was caused by movement of the substance of a given muscle-fiber rather slowly toward a given region, where it became piled up in an area much broader than the width of the muscle fiber. The cross-striations in this region were nearer together than were those in other parts.of the fiber. There was no question of the fact that the material of the fiber moved toward this region, for a granule or a foreign bod.y upon such a flowing fiber finally reached and became included within the broader area. The fiber became stretched out at the end from wliich the protoplasm was flowing, so that the cross-striations in this region became extended far apart. In some cases, while the movement of the protoplasm was taking place, the direction of the current was suddenly reversed, so that the flow occurred in the opp(jsite direction. This lasted only a very short period of time but caused all sorts of cUstortion of the cross-striations just at the beginning of the broader region (contracted area). In some cases they were drawn out on either side and i)re.scnted an appearance such as that .shown by Schiifer (1912, fig. 286); in others there resulted a tearing of the region adjacent to the contracted area which caused the formation of a homogeneous one resembling that depicted by Meckel. In a number of cases the distortion appeared to be cau.sed by the uneven

MUSCULAR CONTRACTION IN TISSUE-CULTURES. 207

liull of the reversed current. Schafer attributes the homogeneous area to a longitudinal shifting of the fibrils, due to their being unequally pulled upon by the contracted part.

So far as I have been able to determine, either in these preparations of sUces of muscle fiber or by a study of the muscular tissue of the living copepod, myofibrils were not exliibited as definite threads in the cross-striated muscle until some change, resulting in the death of the muscle, had occurred. In places where the muscle fiber was not greatly stretched during contraction the reversal of the cur- rent did not take place. In such cases the contracted area remained as it was, without either a homogeneous area or an area of distortion between the contracted region and the remainder of the fiber. Such figures have been described by other observers (Flogel, 1872; Hurthle, 1909, etc.). This same phenomenon of contrac- tion waves, with or without the reversed current, were clearly observed in the muscles of the Uving copepod after experimentation, but it did not resemble the twitch exhibited by the leg muscle of the same animal during normal contraction. In addition to this, the stimulus which brought it about alwaj's resulted in the death of the copepod. The piling up of the muscular material in an area which is broader than the muscle fiber, and in which the cross-striations are closer together, is in all probability, as has been shown by Schafer (1891a), an expression of the phenom- enon of rigor mortis.

DISCUSSION.

Among the muscle cells of tissue-culture growth, whether originating from the amnion, from the heart, or from the skeletal muscles, there are always to be found isolated embryonic muscle cells capable of contraction. The cytoplasm of these cells does not contain any characteristic structure, but is marked by a higher refraction than that of other kinds of cells. The contraction exhibited by each muscle cell is, as above shown, characteristic for the type of tissue from which it arises. In spite of this, however, the actual process is the same for the different types of muscular cells — i. e., there is a point somewhere within the protoplasm of the cell at which some change takes place, drawing the protoplasm towards this region, and resulting in the shortening and thickening of the area involved. A neutralization of the active change then occurs, accompanied by relaxation or a return of the protoplasm to its normal position.

These observations can have Uttle or no bearing on the problem of the causes of muscular contraction without definite phj'siological experimentation. However, they may throw some further light upon a few of the theories previously advanced. For instance, the change can scarcely be due to the imbibition of water at the point where contraction is exhibited (INIcDougall, 1897; ^leigs, 1908), for in that case there would probably be no currents of protoplasm toward the point at which the change takes place. Since myofibrils can not be demonstrated in these Uving cells, the activity can not be based upon the increase of fluid within a definite column such as myofibril (Roule, 1890; Imbert, 1897). The observation of Holmgren (1910) that there is a change of substance from the granules of the cell into the myofibrils is not confirmed, since no such change occurred in the

208 MUSCULAR CONTRACTION IN TISSUE-CULTURES.

granules present in these cells, nor were myofibrils observed. Pseudopodia were formed b}' the cell regardless of the fact that it was undergoing rhythmical contraction; so that while there is a change in the ])o.sition of the protoplasm during contraction, it is not dependent solely ujion those factors which are involved in the formation of pseudopodia (Verworn, 1895).

A few experiments with various fats and soaps were suggested by Quincke's theory of protoplasmic motion. These showed that, while it was possible to cause the formation of pseudopodia (?'. e., blebs of clear protoplasm) this change in the position of a given portion of the protoplasm was not accompanied by contraction of the cell. One of these experiments was as follows:

A minute drop of oleic acid was placed in the medium surrounding an amnion smooth-muscle cell. This caused the withdrawal of the intercellular bridges, so that while the cell remained more or less spread out, it appeared as a somewhat elongated, granular cell with knobs of non-granular protoplasm along the sides. From the region of the knobs blebs of clear cytojilasm, which appeared to be much more fluid than that of a normal cell, were rapidly extended and retracted, only to form again in the same or another region. This activity continued for several hours. Frequently, as many as four or five such areas of bleb formation were observed along the surface of a given cell. The protrusion of the blebs, notwith- standing their large size in some cases, was not accompanied by a shortening of the cell, nor was a current of protoplasm induced towards the region from which the bleb was extruded. This activity, while interesting in itself, did not in any manner resemble that of contraction.

In regard to the theory that surface tension plays a part in the phenomenon, the following experiment is rather interesting. Certain inactive amnion smooth- muscle cells were touched with a delicate glass bristle.^ The cell touched immedi- ately drew together, causing numerous swellings or folds to form on its surface. These resemble the blebs described during mitosis of the cell (Lewis and Lewis, 1917). The folds shortly disappeared, after which rhj'thmical contraction was initiated and continued to be exhibited for some time. However, this is but another way of stating that mechanical stimulation induced contraction.

Changes in the osmotic pressure of the medium, such as were suggested by the experiments of Beutner (1913), did not sufficiently influence the contraction of the muscle cells in cultures to enable one to draw any definite conclusions. No results were obtained such as would show that the jirocess taking place during contraction of the muscle cells in cultures is comparable to that supposed bj' Lillie (1901) to take jjlace during the movement of the cilia of certain ctenaphores. Rhythmical contractions were exhibited for many hours by the muscle cells in tissue cultures, but it is impossible to estimate what changes may have occurred in the medium immediately surrounding the cell, due to the movements of the cell itself or to chemical interchanges, such as between the air and the medium, waste products and the solution, etc.

From the above observations it might almost l)e inferred that it is a normal procedure for embryonic muscular tissue to undergo contraction. While in the

' This experiment was performed by Dr. Robert Chambers.

MUSCULAR CONTRACTION IN TISSUE-CULTURES. 209

organism this activity becomes controlled by various influences, such as the nervous system, the differentiation of the cells, etc., yet when this tissue is removed from these controUing elements, as in tissue-cultures, the muscle cell again undergoes contraction as a part of its normal hfe processes. However that may be, the fact remains that in tissue-cultures the muscle cell, whether originating from the amnion, the heart, or the skeletal muscle, may exliibit this property.

CONCLUSION.

Contraction, characteristic of the amnion smooth muscle, the heart muscle, and the skeletal muscle, takes place in the growth from these muscles in tissue cultures in Locke-Lewis solution.

The phenomenon of contraction, while characteristic for a given type of tissue, is not dependent uj^on a complicated muscular structure such as myofibrils.

The mechanism for the studj^ of the phenomenon of contraction may be reduced to a single, shghtly differentiated, living cell in Locke-Lewis solution.

BIBLIOGRAPHY.

Barfurth, D., ISOl. Ueber Zellbriickon glatter Mus-

kelfftsorn. Arch. f. mikr. Anat., Bd. 38. Benda, C, 1902. Ucber den feineren Bau der glatten

Muskelfasern dos Menschen. ' Verb, der anat.

Gesellschaft, p. 214. Beutner, R., 1913. Einige wcitere Versuche betreffend

osmotische und kolloidale Quellung des Muskels.

Bioch. Zeitschr., vol. 48, p. 217. BiEDERMANN, 1880. ZuT Lehre voin Bau der querges-

treiften Muskelfasern. Sitz. d. KaLs. Akad. d.

Wiss. Wien, Bd. 82. Bi'RHOWS, M. T., 1912. Rhythmische Kontraktionen

der isolierten Herzmuskelzelle au-sserhalb des

Organismus. Milnch. med. Wochensch., Bd. 27,

p. 1473. BuT.scuLi, 1892. Untersuchiingen uber microscopische

Schilume und das Protoplasma. Leipzig. Cii.\MBERs, R., 1917. Microdissection studies. Jour.

Exper. Zool., vol. 23, p. 483. Champy, C., 1914. Quelques rSsultats de la m6thode de

culture des tissues. Le muscle lisse. Arch, de

Zool. Exp6r. et Gen^rale, Notes et Revue, t. 53, p.

42. CoNGDON, E. D., 1915. The identification of tissues in

artificial cultures. Anat. Rec, vol. 9, p. 343. CoWDKY, E. v., 1914. The comparative distribution of

mitochondria in spinal ganghon cells of verte- brates. Amer. Jour. Anat., vol. 17, p. 1. DoBROWOLSKT, N. A,, 1916. Sur la culture des tissus

des poissons et d'autres animaux inf 6rieurs. Comp.

rend de la Soc. de Biol., t. 79, p. 789. Ddval, M., 1889. Atlas d'Embryologie, p. 14. Engelmann, T. \V., 1869. Zui' Physiologic des Ureter.

Arch, fiir d gesammte Physiol., Bd. 2, p. 243. , 1875. Ueber den faserigen Bau der kontraktilen

Substanz mit, etc. Arch. f. ges. Physiol., Bd. 11,

p. 432. , 1893. Ueber den Ursprung der Muskelkiaft.

Leipzig. Floqel, J. H., 1872. Ueber die quergestreiften Muskeln

der Milben. Arch. f. mikr. Anat., Bd. 8, p. 69. FoRSTER, E., 1904. Die Kontraktion tier glatten Mus-

kelzellen und der Herzmuskelzellen. Anat. Anz.,

Bd. 25, p. 338. FuLLEBORN, F., 1895. Beitrage zur Entwickelung der

AUantois der Vogel. Diss. Berlin. Garrey, W. E., 1905. Twitching of skeletal muscle

produced by salt solution with special reference

to twitching of mammaUan muscles. Amer.

Jour. Physiol., vol. 13, p. 3. Gaskell, W. II., 1S82. On the rh>'thm of the heart of

the frog. Croonian Lecture. Phil. Trans., p. 993. , 1883. On the innervation of the heart, etc.

Jour. Physiol., vol. 4, p. 43. Godlewski, E., 1902. Die Entwickelung des Skelet- und

Herzmuskelgewebes der Saugethiere. Arch. f.

mikr. Anat., Bd. GO, p. 111. Green, C. N., 1899. On the relation of the inorganic

salts of blood to the automatic activity of a strip

of ventricular muscle. Amer. Jour. Physiol.,

vol. 2, p. 82. GrCtzner, p., 1904. Die glatten Muskeln. Ergebn. d.

Physiol., II Abth., Bd. 3, p. 12. Harrison, R. G., 1910. The outgrowth of the nerve

fiber as a mode of protoplasmic movement. Jour.

Exp. Zool., vol. 9, p. 877.

Holm(;hen, Emil, 1910. T^ntersuchungen uber die morphologisch nachweisbaren stofflichen Umset- zungen der quergestreiften Muskelfasern. Arch. f. mikr Anat., Bd. 75, p. 240.

Hooker, D. R., 1912. The effect of carbon dioxide and of oxygen upon muscular tone in the blood ves- sels and ahmentary canal. Amer. Jour. Physiol., vol. 31, p. 47.

Howell, W. H., 1898. The relation of the blood to the automaticity and sequence of the heart beat. Amer. Jour. Physiol., vol. 2, p. 47.

Hi'iRTHLE, K., 1909. Ueber die Struktur der querges- treiften Muskelfasern von Hydrophilus im ruhen- den und tatigen Zustand. Arch. f. d. ges. Physiol., Bd. 126.

Imbert, a., 1897. Le mdchanisme de la contraction musculaire. Arch, de Physiol., t. 9, p. 289.

Krause, W., 1873. Die Kontraktion dor Muskelfaser. Arch. f. d. ges. Physiol., Bd. 7.

Lake, N. C., 1916. Observations upon the growth of tissues in vitro, etc. Jour. Physiol., vol. 50, p. 364.

Langley, J. N., 1908. Nicotine on frog's arm muscle. Jour. Physiol., vol. 37, p. 165.

, and R. Magnus., 1905. Some observations on

the movement of the intestine, etc. Jour. Physiol., vol. 33, p. 34.

Lenhossek, M., 1899. Das Mikrocentrum der glatten Muskelzellen. Anat. Anz., Bd. 16.

Levi, G., 1916a. II ritmo e le modality della mitosi nelle cellule viventi coltivate in vitro. Arch. Ital di. Anat. e di Emb., vol. 15, p. 243.

, 19166. Migrazione di elementi specifici difTeren-

ziati in culture di miocardio e di muscoh schele- trici. Arch. p. 1. Scienze Medichi, vol. 40, p. 1.

Lewis, M. R., 1915. Rhythmical contraction of skeletal muscle in tissue cultures. Jour. Physiol., vol. 38, p. 153.

, 1916. Sea water as a medium for tissue cultures.

Anat. Rec, vol. 10.

Lewis and Lewis, 1912a. Membrane formation from tissues transplanted in artificial media. Anat. Record, vol. 6, p. 195.

, 19126. The cultivation of sympathetic nerves

from intestine of chick embryos in saline solution. Anat. Rec, vol. 6, p. 7.

, 1915. Mitochondria and other cytoplasmic

structures. Amer. Jour. Anat., vol. 17, p. 339.

, 1917a. The contraction of smooth muscle cells

in tissue cultures. Amer. Jour. Physiol., vol. 44, p. 67.

, 19176. Behavior of cross-striated muscle in tis- sue cultures. Amer. Jour. Anat., vol. 22, p. 109.

, 1917c. The duration of the various phases of

mitosis in the mesenchyme cells of tissue cultures. .\nat. Rec, vol. 13., p. 359.

Lewis, M. R., and W. R. Robertson, 1916. The mi- tochondria and other structures, etc. Biol. Bull., vol. 30, p. 99.

Leydig, 1849. Zur Anatomic von Piscicola geometrica. Zeitschr. f. wi.ss. Zoijl., Bd. 1.

LiLLiE, R., 1901. The relation of ions to contractile processes. Amer. Jour. Physiol., vol. 21, p. 200. LE, D. T., 1901. The action of certain ions on ven- tricular muscle. Amer. Jour. Physiol., vol. 4, p. 265. — , 1903. The importance of sodium chloride in heart activity, .\riier. .hmr. I'liysiol., vol. S, p. 75.

LlNGl

210

BIBLIOGRAPHY.

211

LOEB, J., 1899. Festschrift fur Prof. Fiok. Wurzburg.

, 1906. Dynamics of LiviiiK Mutter. New York.

McDouGALL, W., 1897. On the structure of cross-striated muscle and a suggestion as to the nature of its contraction. Joiu-. Anat. and Physiol., vol. 31 , p. 410.

McGiLL, C, 1907a. The histogensis of smooth muscle in the ahmentarj' canal and respiratory tract of the pig. Intern. Monatschr. f. Anat., Bd. 24, p. 209.

■ ■ — , 19076. The syncj-tial structure of smooth mus- cle. Anat. Rec, vol. 1, p. 91.

, 1909. The structure of smooth muscle in the

resting and in the contracted condition. Amer. Jour. Anat., vol. 9, p. 493.

, 1910. Studies on sjnooth muscle. Quart. Journ.

Exper. Physiol, vol. 3, p. 241.

M.\GNus, R., 1904. Versuche am iiberlebenden Diinn- darm von Siiugethieren. Arch. f. d. ges. Physiol., Bd. 102, p. 123.

Marinesco, G., 1912. Forschungen iiber den kolloideu Bau der NervenzeUen und ihre erfahrungsge- miissen Veranderungen. Kolloid-Zeitschr., Bd. 9, p. 209.

Marsh.all, C, 1887. Observations on the structure and distribution of striped and unstriped muscle in the animal kingdom and a theory of muscu'ar contraction. Quart. Joiu-. of Mic. Soc, vol. 28.

Meek, A., 1898. Preliminary note on the post embiy- onal history of striped muscle fibre in mammals. Anat. Anz., Bd. 14, p. 619.

Meigs, E. B., 1908. The structure of the elements of cross-striated muscle and the changes of form which it undergoes during contraction. Zeit- schrift f. aUg. Physiol., Bd. 8, p. 81.

Merkel, Fr., 1872. Die quergestreifte Muskel. Arch, f. mikr. Anat., Bd. 8, p. 244.

Mines, J. R., 1908. On the spontaneous movement of amphibian skeletal muscle in saline solutions, etc. Jour. Physiol., vol. 37, p. 408.

Moore, A., 1901. Are the contractions of the lymph heart of the frog dependent upon centers situated in the spinal cord? Amer. Jour. Phys., vol. 5, p. 196.

Mott, F., 1912. The bio-physics and bio-chemistrj' of the neurone. Brit. Med. Jour., p. 780.

Quinxke, 1888. Theorj' of protoplasmic motion quoted from J. Loeb, 1906 : Dynamics of Living Matter, p. 55.

Ranvier, 1880. Legons d'anatomie gen^rale sur le systeme musculaire, Paris.

Remak, 1861. Ueber den Bau und die Zusammenset- zung der Muskelfasem. Wiener Sitzungsber., Bd. 44.

Renaut, J., 1893. Traits d'Histologie Pratique Paris, p. 557.

Rohde, E., 1905. Untersuchungen ueber den Bau der Zelle. Zeitschr. f. wiss. ZooL, Bd. 78, p. 1.

Rollett, a., 1886. Bau des Quergestreiften Muskel- fasem. Wiener Denschriften, Bd. 51.

Roth, G. B., 1907. Movement of excised ureter of dog.

Amer. Jour. Physiol., vol. 44. Rocl£, L., 1890. Ktude sur le dCveloppement et la

structure du tissu musculaire. Thesis, Paris. Sabin, F. R., 1917. Origin and development of the

primitive vessels of the chick and of the pig.

Contributions to Embryology, vol. 6. Pub. 226,

Carnegie In.st. Wash., p. 61. Schafer, E. a., 1873. On the minute structure of the

leg muscle of the wat<;r beetle. Phil. Trans.,

vol. 163, p. 429. , 1891a. On the structure of cross-striated

muscle. Internat. Monatschr. f. Anat. u Physiol.,

Bd. 8, p. 177. , 18916. On the minute structure of the muscle

columns which form the wing muscles of insects.

Proc. of Roy. Soc, vol. 49, p. 281. , 1910. On McDougall's theory of muscular

contraction, etc. Quart. Journ. Exper. Phvsiol.,

vol. 3, p. 63. , 1912. Microscopic anatomy. Quain's Anat- omy, p. 195. ScHAFFER, J., 1899. Zur Kenntnis der glatten Mu.skel

zellen, insbesondere ihrer Verbindung. Zeitschr.

f. wiss. Zoo!., Bd. 66, p. 214. ScHOCKAERT, A., 1909. NouveUes recherches compara- tives sur la texture et le developpcment du myo-

carde chez les vert^bres. .Arch, de Biol., t. 24. ScHULTZ, P., 1895. Die glatte Musculatur der Wir-

belthiere. Arch. f. Anat. u. Physiol., Phvsiol.

Abth., p. 517. ScHWALBE, G., 1868. Beitrage zur Kenntnis der glatten

Muskelfasem. Arch, f . mikr. .Anat., Bd. 4, p. 392. Shipley, P. G., 1916. The development of erj-throcj-tes

from hemoglobin-free cells and the differentiation

of heart fibers in tissue cultivated in plasma.

Anat. Rec, vol. 10, p. 347. Soli, Ugo, 1906. Sulla struttura delle fibre muscolari

liscie dello stomaco degli uccelli. Anat. Anz.,

Bd. 29, p. 586. Stiles, P. G., 1901. On the rhythmic activity of the

oesophagus and the influence upon it of various

media. Amer. Jour. Physiol., vol. 5, p. 338. SuNDWALL, J., 1912. Tissue proliferation in plasma

medium. Bull. U. S. Hyg. Lab. and Mar. Hosp.,

vol. 81. V.AN Gehuchten, a., 1889. Les noyaux des cellules

musculaires striees. Anat. .Anz., Bd. 4. Verworn, M., 1895. AUgemine Physiologic, Jena. Verz.ar, F., 1908. Ueber die Anordung der glatten Mus-

kelzellen im Amnion des Huhnchens. Intern.

Monatsschrift f. Anat., Bd. 24, p. 292. Wagexer, G. R., 1867. Die Entwicklung und den Bau

der quergestreiften und glatt«n MuskeKasem.

Sitz. d. Gesell z. Beford d. ges. nat. z. Marburg,

Bd. 10, p. 82. Werner, G., 1894. Zur Histologic der gktten musku-

latur. Diss. Jurjew.

DESCRIPTION OF PLATES.

Plate 1.

F,(j. 7. — ^I'hotossraph of a fixed preparation of the growth from the heart of a 6-day chick embryo. Culture 48 hours

old. Oc. 4, lens 16 mm. Fia. 8. — ^Photoxraph of a fixed preparation of the growth from the skeletal muscles of a 9-day chick embryo. Culture

4S hours old. Oc. 4, lens, 10 mm. p,Q 9. — Photograph of a fixed preparation of the growth from the amnion of a 5-day chick embryo. Culture 48

hours old. Oc. 4, lens 10 mm. Fio. 10. — Photograph of the protoplasmic end of a muscle sprout. The contractile suKstance is coagulatinl in the

form of bualles of threads. Culture 4S hours old, from skeletal muscle of 8-day chick embryo. Fig. 11. — Cross-striated muscle fibers in growth from skeletal muscle of a 10-day chick embryo. Culture 5 days old.

Fixed by means of Zenker's solution without acid. Stain iron-hematoxylin. Oc. 6, lens 2 mm.

Plate II.

Fio. 12. — An epithelial cell lyin-^ side by side with the smooth-muscle cell of fig. 17. It is not elongated, does not

contain coagulated contractile substance, and the mitochondria are not long threads. Fig. 13. — Three cells observed while undergoing rythmical contraction and later fixed by means of Zenker's solution.

C, the contraction node. Culture 25 hours old, from amnion of 8-day chick embryo. Oc. 4, oil-imm. Fig. 14. — Smooth muscle from the amnion of an 8-day chick embryo. Culture 48 hours old. FLxed with 10 per cent

nitric acid. Stainefl with Mallory's connective-tissue stain. Curled edges of cell stain jis eliistic fibers,

while the coagulated remains of intercellular bridges appear as a connective-tissue network. Oc. 4, oil-imm. Fig. 15. — The star-shaped arrangement of the muscle fibers of the amnion of .5-day chick embryo. Oc. 0, lens 16 mm. Fig. 16. — A group of smooth muscle-cells from the amnion of a 4-day chick embryo. Culture 48 hours old. The

contractile substance is coagulated into bundles of gray threads. Osmic vapor, iron hematoxylin

and cosin. Oc. 6, lens 3 mm. Fig. 17.— Sprea<l-out smooth-muscle cell from the edge of the growth. The coagvdated contractile material can be

distinguished from the elongated mitochondria. Culture 48 hours old from 5-day chick embryo.

Oc. 4, oil-immersion lens.

212