Protoplasmic Action and Nervous Action

Recent observations by Just^ have emphasized still more fully the resemblance between the primary or surface change in the activation of egg cells and in the stimulation of irritable tissues. In the large egg of the sand-dollar, Echinarachnius (ca. 140 ju in diameter), the first visible effect of insemination is an alteration of the egg surface, beginning at the point of entrance of the spermatozoon. A liquefactive or secretory change occurs in the protoplasmic surface layer, in consequence of which a thin surface-film is separated to form the fertilization membrane; this process of separation is not simultaneous at all points on the surface, but progresses in a wavelike manner from the point of sperm-entry to the opposite pole, which it reaches about 20 seconds later (at 20°). During the propagation of this disturbance over the egg surface, the latter is altered in such a manner that the plasma membrane loses temporarily its normal tenacity and coherence; this effect is readily demonstrated by placing the eggs at this time in dilute sea water (60 vols, fresh plus 40 sea water) in which they undergo immediate and rapid disintegration. The breakdown of the surface layer can be seen to begin at the region where the fertilization membrane is beginning its separation. This period of instability lasts only for the brief period (about one minute) during which the ''cortical reaction" is travehng over the egg surface; within about a minute after insemination the original resistance to dilute sea water has returned, showing a restitution of the normal coherent surface layer. Thus a characteristic surface-change, apparently accompanied by a local disintegration or disorganization of the plasma membrane, constitutes the first reaction of this egg to fertilization; this change is propagated as a wave over the cell surface and is followed by a reconstructive process restoring the original condition. As we have already seen, there are indications that a propagated surface-change of a similar kind, only with different time-

relations and a different velocity of propagation, accompanies stimulation in irritable cells and nerve fibers. During the formation of the cleavage-furrow in celldivision a similar reversible change in the physical consistency and coherence of the cell surface occurs in echinoderm eggs;^ and there are many indications that the same kind of change is of general occurrence in dividing cells. The case of cell-division is of special interest, since a rhythm of chemical change and of susceptibility to physical and chemical injury is also associated with the rhythm of the cleavage-process. Lyon's experiments indicate that at the time when the cleavage-furrow is forming in the Arbacia egg, the rate of evolution of CO2 is several times greater than in the intervals between cleavage;^ at this time the egg is also most susceptible to injury by heat, ultra-violet radiation, deprivation of oxygen, and poisons (KCN, acids, and organic compounds). The time-relations of the accompanying change in the plasma membrane can be followed readily and accurately by transferring successive portions of a single lot of recently fertilized Arbacia eggs (in which the cleavage-process is very regular and occurs simultaneously in all eggs) from normal sea water to dilute sea water (50 to 60 volumes fresh water in 100 of the mixture) at regular intervals before, during, and after the formation of the cleavage-furrow.^ Eggs which are thus treated some time before cleavage swell osmotically but without undergoing evident increase of permeabihty

or losing the power of development on return to sea water; at about the time when the furrow begins to form, there is a marked and rapid decline of extensibihty and coherence in the plasma membrane, and the eggs show rapid loss of pigment and cytolysis when transferred to the dilute sea water. Eggs brought into dilute sea water a few minutes after the furrow is complete are found to have recovered the original resistance, and swell without cytolysis. These experiments show clearly that accompanying the division of the cell body there is a reversible change in the properties of the plasma membrane, this change involving both loss of coherence and increase of permeabiHty. That the permeabihty as well as the physical tenacity of the membrane is altered is best shown by studying the behavior of the eggs in concentrated instead of dilute sea water; during the formation of the furrow the abstraction of water and shrinkage are distinctly less rapid and complete than before or after cleavage, a difference indicating a partial loss of semi-permeabiHty at this time. Other indications of increase of permeabihty during cleavage have been noted by various observers (Harvey, Just, Lyon).

According to the present theory variations of electrical surface-potential should accompany these changes of permeabihty; and observations made at Woods Hole in 1904 by Miss Hyde,^ using fish eggs, indicate that during the formation of the cleavage-furrow the blasto-j disk area becomes increasingly negative relatively to the] general surface of the egg. Experiments in this field are, however, few in number as yet; and it would bei desirable to repeat and extend these observations, using |

the thermionic ampHfier to enhance the minute effects obtainable from single eggs, and the string galvanometer as the recording instrument. It should be noted, however, that Miss Hyde's observations are in conformity with those of other investigators who have found rapidly growing regions of plants and animals — ^i.e., those where cell-division is in active progress — ^to be electrically negative to more slowly growing regions.^ Evidence that reversible variations of permeability are associated with such processes as fertihzation and celldivision may seem to have a somewhat indirect bearing on the problem of the conditions of stimulation in tj^ical irritable tissues like muscle and nerve. Yet all of these vital processes are alike in being subject to initiation or control by environmental conditions or events; in other words, they all illustrate the characteristic ^'irritabihty" of living matter. The fundamental conditions determining and controlHng the metabolic reactions which furnish the energy for vital processes are in all probability everywhere the same. Hence the above-cited facts indicating that changes of permeability are regular accompaniments of fertilization and cell-division are confirmatory evidence for the view that such changes play an essential part in other manifestations of irritabihty. In all forms of protoplasm the essential metabolic reactions occur under the control of the film-partitioned or emulsion-Uke structure of the living system; it is therefore to be expected that they will vary in their rate and character

with alterations in this structure. In particular the foregoing evidence indicates that the temporary breakdown of the semi-permeable surface-lamella or limiting layer of the protoplasmic emulsion influences profoundly the chemical and other processes occurring in the cell interior; apparently the effects of this surface-change are transmitted throughout the whole mass of protoplasm, and the physiological activities of the cell are changed correspondingly.

It has already been mentioned that experiments of a kind closely analogous to those described for unfertilized egg cells may be performed with irritable tissues like muscle and nerve, both of which when immersed in pure isotonic solutions of neutral sodium salts undergo rhythmical or other stimulation, which may be checked by calcium salts or anaesthetics; and the above-cited experiments with Arenicola larvag afford other and more direct evidence that stimulation and permeabilityincrease are closely associated. Hober's experiments on the influence of anaesthetics in checking the development of the negative electrical variation produced in muscle by application of KCl solution furnish evidence of a similar kind. The effects of neutral salts may thus be more or less completely antagonized by anaesthetics as well as by the alkali earth cations. In all of these cases prevention of permeability-increasing action runs parallel with prevention of stimulation or of the normal manifestations of stimulation.^

Conversely any strongly cytolytic action has a stimulating effect. The larvae of Arenicola contract strongly in solutions of cytolytic agents Uke chloroform; this effect is irreversible and is associated with a marked increase of permeability and rapid death. Phenomena of a similar kind are seen in vertebrate skeletal muscle. The permanent or irreversible shortening or '^ contracture" of frogs' muscle in solutions containing cytolytic substances, e.g., saturated solution of chloroform in Ringer's solution, is well known; this contraction is associated with a large production of lactic acid, and apart from its irreversible or ^' rigor" character bears many resemblances to normal contraction. A similar contraction accompanies the onset of heat-rigor and other forms of death-rigor, and in all such cases the structure of the cells is profoundly altered, the permeability undergoing marked increase while the fibrils lose their tensile strength and elasticity. These changes have a close general resemblance to those already described as accompanying the accelerated rhythmical activity—^ indicating excessive stimulation — -of the ctenophore swimming plate in pure Na salt solutions.

The foregoing contraction-producing action of cytolytic substances on frog's muscle may be made to resemble more closely the phenomena of normal stimulation by first ''sensitizing" the muscle by irnmersing it for a few minutes in a pure isotonic solution of a neutral Na salt (NaCl, NaBr, Nal, NaN03, etc.). The fresh isolated gastrocnemius (normal or curarized), immersed in Ringer's solution and arranged so as to write upon a smoked drum, is transferred for four or five minutes to the pure solution of the Na salt, from which it is brought directly into the solution containing the cytolytic

substance. The contraction then resulting is much more rapid and vigorous than in the control muscle which is brought into the same solution directly from Ringer; the degree of permanent shortening is also greater, as is also the degree of coagulation of the muscle protoplasm, as shown by the whitening or opacity produced. The action of contraction-producing salt solutions Uke KCl, Na tartrate, sulphate, and citrate is similarly intensified by a previous bath of the kind above; also the rapidity of onset of heat-rigor, with the associated contraction, when the muscle is dipped in warm Ringer's solution (38'-40°). These sensitization-effects have been demonstrated with the following cytolytic substances: chloroform, cytolytic glucosides (saponin, digitahn, aconitin, and agaricin), tetanus toxin, rattlesnake venom, foreign blood sera (horse, dog), and soaps. The degree of the stimulation following the introduction of the saltsensitized muscle into the solution, as indicated by the rate and degree of the contraction, is in general proportional to the intensity of the cytolytic action (as shown by varying the concentration and nature of the cytolytic substances) .^

Since in all such experiments the contraction follows immediately (within a second or less) after placing the muscle in the stimulating solution, there seems to be no doubt that the initiatory effect consists in an alteration of the external surface layer of the muscle cells. Apparently, when the normal muscle is exposed to the pure salt solution, the cell surface is rendered more susceptible to alteration by external chemical agents, and the susceptibility to chemical stimulation of the kind above

is correspondingly increased. The relative insusceptibility of the normal muscle depends on the presence of Ca salts in the external medium; if, instead of a pure solution of the sensitizing salt, one containing CaClz (i mol CaCla to 20 Na salt) is used, no such effects are obtained.' The sensitizing action is thus subject to typical salt-antagonism, like so many other biological processes, especially those involving alteration of the protoplasmic surface layers. Muscles which have been rendered hypersensitive by exposure to the pure salt solution, rapidly recover their normal properties on return to Ringer's solution. The inverse type of effect, decrease of susceptibiHty to chemical stimulation, may be induced by a similar exposure to isotonic solutions of CaClz, MgClz or similar salts. Such desensitizing effects are closely related to those classed under narcosis, depression, or anaesthesia, and are also reversible in Ringer's solution.

A related type of salt sensitization, produced by isotonic solutions of Na salts whose anions precipitate calcium (or remove Ca ions from solution) , was described by Loeb in 1901.^ Muscles dipped for a few minutes in solutions of Na sulphate, tartrate, citrate, or similar salt, and then brought into the air (or other foreign medium, e.g., oil), exhibit vigorous tetanic contractions, which cease or are diminished on return to the salt solution. Apparently this reaction depends on an altered contact-sensibility, due to some modification of the cell surface. It illustrates a type of effect which appears to be widely prevalent in irritable elements.

1 Unpublished observations in the Biological Laboratory of Clark University. Nerve is affected similarly; and the characteristic pharmacological effects produced by this group of salts, e.g., their cathartic action (which apparently depends upon a heightening of contact-irritabihty in the intestinal tract), are probably referable to conditions of a similar kind. There is much evidence that many forms of pharmacological action are due to changes in the physical consistency, permeabiHty, chemical alterability, or other properties of the protoplasmic surface-films.

It is important to note the relation of sensitizations of the class described— which consist in a general heightening of irritability toward non-specific chemical or other stimulating conditions — to the class of specific sensitizations, of which anaphylaxis is the most striking example. The general features of this phenomenon are well known. During the early stages of the process of immimization, following the introduction of a foreign protein into the circulation, the cells of the mammahan organism become highly sensitive to the introduction of further protein of the same kind, and in certain animals, notably the guinea-pig, the most conspicuous effect of the second injection is seen in the smooth muscle cells, especially those of the respiratory tract; these contract firmly and persistently and occlude the bronchioles, with death by asphyxiation as a consequence. This contraction-producing effect is almost certainly dependent on a specific antigen-anti-body reaction occurring in the surface layer of the muscle cells. The promptitude with which it follows injection of even a small quantity of the foreign protein indicates this, since the latent period seems insuf&cient for penetration into the cell interior; such a conclusion receives further support

from the facts of passive sensitization, in which the sensitized condition is produced in a normal guinea-pig by injection of blood from another animal which has already been immunized to the protein in question. Passive sensitization can also be produced in vitro by bathing strips of uterus with serum from an immunized animal. The most probable interpretation of this phenomenon is that the circulating anti-body is adsorbed or fixed by contact with the smooth muscle cells, thus becoming a constituent of the protoplasmic surface layer. When the antigen (the original protein used for immunization) is introduced, it reacts with the adsorbed anti-body, and in so doing alters the structure or consistency or permeabiHty of the surface layer (very much as a specific cytolysin would do) in a manner corresponding to strong stimulation. Contraction then results; and since the antigen-anti-body reaction is an irreversible one, the muscle cells remain firmly and persistently contracted, with results fatal to the animal. Dale has brought forward evidence that the specific chemical interaction underlying anaphylactic shock is identical with the precipitin reaction, the difference being that the reaction occurs within the cell instead of in the blood stream.^ If this is the case, it is easy to understand why powerful stimulating effects should result from precipitation of proteins within the protoplasmic surface-film, since such a process must alter the structure and permeability of this layer and hence act as a stimulating condition in the same manner as any other cytolytic change would do.

^ Dale, Croonian Lecture, Proceedings of the Royal Society, B, XCI According to this conception the anaphylactic reaction of smooth muscle cells becomes an example of specific chemical stimulation, dependent on the presence of specific substances (presumably protein) in the surface layer of the reacting cell; these substances react with substances of corresponding or complementary configuration in the environment, and hence furnish the conditions for a highly selective and specific type of response. Presumably other forms of specific chemical sensitivity are also to be referred to the presence of specific chemical compounds in the surface-films of the reacting cells. Such a conception renders clearer the general nature of the relation between the special chemical sensitivity exhibited by a particular species of cell and its specific chemical organization. Any change in the chemical constitution or physical state of the plasma membrane must influence irritability and hence the other properties or activities controlled by this region of the cell.

Certain instances of stimulation-effects which are associated with an evident increase of surfacepermeabihty have already been cited. Perhaps the clearest instance of a direct dependence of a normal functional response upon a sudden increase of permeability is seen in the osmotic motor mechanisms of plants, such as the Venus' flytrap and the sensitive plant. In the latter plant, Mimosa pudica, the leaves are kept in the normal expanded and upright position by turgid or water-distended masses of parenchyma cells (pulvini)

at the base of each leaflet and petiole. This turgor, as in other herbaceous tissues, is maintained by the osmotic pressure of the cell-contents; this pressure, acting against the semi-permeable plasma membranes, causes the entrance of water from the intercellular spaces and distends the cells until the pressure is equihbrated by the elastic tension of the stretched cellulose cell walls. Evidently the continued maintenance of this condition depends on the preservation of semi-permeability. On stimulation there is a sudden loss of turgor, accompanied by exit of water and dissolved substances from the cells; the stretched cell walls of the pulvini contract, the leaves fall, and the leaflets fold together. Apparently stimulation renders the plasma membrane suddenly permeable to the osmotically active intracellular substances which maintain turgor. This effect is reversible, and under normal conditions turgor is gradually regained. The leaves of the Venus' flytrap and the sensitive contractile stamens of the Cynarece show a behavior essentially similar to that of Mimosa. Temporary loss of semi-permeability due to mechanical stimulation seems to be a not uncommon phenomenon in plant cells; Pfeffer cites the ''stimulatory plasmolysis" of diatoms and other plant cells as cases of this kind, although he apparently hesitates to apply this explanation to the pulvinus of Mimosa}

The general rules of stimulation apply to these osmotic motor mechanisms of plants, in the same manner as to the excitation-processes of animal tissues. Electrical stimulation, summation, and anaesthesia occur under conditions similar to those described above, although the quantitative relations are different ; hitherto these relations have been less completely investigated in plants than in animals. Transmission ofexcitation in plants resembles that of slowly conducting animal tissues,' and the excitation-process is accompanied by a negative bioelectric variation. A prolonged refractory period succeeds the motor response in Mimosa, and this condition also appears to be general in plants. The presence of a high degree of turgor in plant cells renders the evidence of a temporary loss of semi-permeability during excitation in many respects more definite and complete than in the case of animal tissues ; but in other respects the fundamental processes underlying stimulation appear to be of the same kind in both groups of organisms.

In the higher animals the phenomena accompanying the secretion of gland cells, especially those under nervous control, show many resemblances to those just described for motile plant tissues. There is the same loss of water and dissolved material from the cell, the same electromotor variation, and the same gradual recovery. The variations in the permeability of the mammalian kidney cells under the influence of fear, excitement, or other abnormal emotional or nervous conditions also suggest that in these cells stimulation is associated with increased permeability; similar evidence is furnished by sweat glands. The secretory phenomena accompanying fertilization in many egg cells have already been mentioned.

It might be objected that evidence drawn from the observation of special tissues whose normal function consists in the separation of dissolved substances, either formed within the cells or collected from the surroundings, is scarcely applicable to the case of irritable elements in general. But such facts at least show plainly that stimulation is often associated with increased permeability or other evidence of temporary structural breakdown; and the fact that the conditions under which stimulation occurs in other irritable living systems, and also its most general manifestations such as the bioelectric variations, are of the same kind in the turgor-motor cells of plants and in gland cells as in muscle and nerve points clearly to the existence of some fundamental physico-chemical condition common to all such promptly reacting irritable systems. If, as the present theory holds, this condition consists in the temporary alteration or breakdown of the film-structure which surrounds and pervades all protoplasmic systems, the resemblances are intel- Kgible; while in any case the differences are to be attributed to special peculiarities of structure and organization.

The phenomena of luminescence in animals furnish additional evidence that stimulation is associated with the temporary breakdown or removal of semi-permeable partitions within the Hving system. The production of light in irritable luminescent organisms like Noctiluca may be regarded as an index of stimulation in very much the same sense as the bioelectric currents are such an index ; and probably both phenomena are conditioned by structural changes of a similar kind. The investigations of Dubois and Harvey indicate that in many if not all luminescent animals light-production depends on the union of the two photogenic components, luciferin and luciferase, in the presence of oxygen.^ In a lumi-

^ Cf. Harvey's recent book, The Nature of Animal Light (Philadelphia, 192a). nescent cell which responds to stimulation by a flash of light, all three substances are apparently present and available, but during the resting state they are prevented from uniting by the presence of protoplasmic films or partitions; when as a result of stimulation these partitions are temporarily broken down, chemical union and light-production result. Further analysis of the conditions of luminescence in irritable cells will no doubt throw much light upon the general nature of stimulation processes.

Harvey^ has recently made some simple and striking experiments on plant tissues, giving further indication that in living cells under normal conditions chemical reactions are frequently prevented or restricted by the presence of protoplasmic partitions or membranes impermeable to the interacting substances. The oxidase reactions which cause the browning of potato, apple, or similar tissues are examples. If a potato is cut in the presence of oxygen, the browning occurs only at the cut surface; even in pure oxygen under high pressure, the interior tissue remains unchanged. This absence of effect cannot be referred to an impermeability to oxygen, since all of the physiological and chemical evidence indicates that living protoplasm is freely penetrated by this gas. The oxidase and the chromogen are in some way prevented from uniting while the tissue is living. If, however, it is exposed for a few minutes to chloroform vapor, the browning extends rapidly throughout the whole mass. Apparently the effect of the chloroform is to break down the protoplasmic partitions which normally prevent free union of the compounds. Destruc-

tion of semi-permeability is a universal effect of poisoning with chloroform or similar substances; this effect is seen in the wilting of turgid plant tissues, increase of electrical conductivity, or diffusion of substances (e.g., coloring materials) from the cells. The leaves of the common false indigo plant, which blacken on death, also afford a striking demonstration.^ If the leaves are poisoned with chloroform in the absence of oxygen (e.g., in a gas chamber with hydrogen) , they remain green ; if they are then exposed to air they blacken immediately. When Hving, intact leaves are exposed to oxygen at 100 atmospheres, no blackening results. On the other hand, mechanical injury, natural death, or poisoning all produce this effect at air tension. The essential condition for the reaction is apparently the destruction of diffusion-preventing partitions which during life keep the interacting substances apart. According to Chiari,^ autolysis of animal tissues is similarly hastened by ether or chloroform. It thus seems probable that in many if not all cells the external layer of protoplasm (plasma membrane) is not the only semi-permeable structure present, but that it is continuous with a system of similarly constituted films pervading the protoplasmic system and determining the spatial distribution of the water-soluble cell-constituents.^

If a temporary breakdown of film-structure can determine chemical effects of this kind, the possibility presents itself that in cells of a different type of organization, e.g., muscle cells, other chemical reactions, including those yielding the energy for contraction, may be under similar kind of control. In these cells the chief reactions following stimulation probably occur at the surface of the contractile fibrils, and apparently certain reactionproducts, e.g., lactic acid, are directly concerned in the resulting contraction.^ We may assume that the temporary breakdown of the interfacial film (between fibril and sacroplasm) will have a double effect: (i) permit access of diffusible substances (possibly of the lactic acid) to the interior of the fibril; and (2) form the condition of a change of surface-tension, in the same general manner as in the Hg-H202 system. Under these conditions contractile effects (essentially of an electrocapillary kind) would result. Hence the consideration of the relation of film-structure to the chemical reactions of protoplasm has an obvious bearing on the problem of the conditions of contractility in muscle and other contractile tissues.

' For a discussion of the part played by acids in the contractile mechanism cf. the recent review of A. V. Hill, Physiological Reviews, II The view that the transmission of the excitationstate from the active region of an irritable protoplasmic element to the adjacent resting region is the result of secondary electric stimulation by the local bioelectric current between the two areas is one which is supported by general theoretical considerations and by a variety of direct and indirect evidence. In a general sense there is nothing novel about this hypothesis, which, like most scientific conceptions, has had its historical background and development; it was expressed tentatively by Du Bois-Reymond^ and in a more definite form by Hermann;'' more recently Kiihne, Cremer, Gotch, Keith Lucas, and others have supported it on various grounds.^ The absence in nerve of any observable accompaniment of the local excitation process, other than the electric variation, which could conceivably serve as a stimulus to the resting region adjoining the active area, is in

^ Gesammelte Ahhandlungen ztir allgemeinen Musket und Nervenphysik, II, p. 698; cf. p. 733. 2 See especially the clear statement by Hermann in his Handbuch, II, 194, cited in Cremer's comprehensive article on nerve physiology in Nagel's Handbuch der Physiologie, IV, 2d half (1909), 929. 3 Kiihne, Croonian Lecture, Proceedings of the Royal Society, XLIV (1888), 446; Cremer, loc. cit.; Gotch, article on nerve in Schafer's textbook, cf. pp. 458, 557 ff.; Keith Lucas, Journal of Physiology, XXXIX

itself a strong argument in its favor. It is surprising that until recently it has received relatively little serious consideration from physiologists, most of whom have been apparently content to regard the bioelectric phenomena as inessential by-products of protoplasmic action. The importance of the electrical factor in the phenomena of protoplasmic transmission is, however, clearly recognized in the ''core-conductor" (Kernleiter) theory or theories of nervous action.^ This conception has as its basis the presence of characteristic polarization effects in a nerve through which a current is led (by non-polarizable electrodes) ; these effects closely resemble those exhibited by a system consisting of a simple mxetallic wire surrounded by a sheath or layer of electrolyte solution. The resemblance is so detailed, as regards the distribution, rate of development, and subsidence of the polarization potentials, that there can be little doubt of the essential identity of the physical conditions underlying these phenomena in the two systems. In nerve the surface of the axone has usually been regarded as the chief seat of the polarization, and Hermann especially has called attention to the intimate relations existing between polarization and stimulation. Neither he nor his successors, however, could explain satisfactorily, on the basis of the phenomena shown by simple polarization models of this type, the characteristic wavelike transmission of the electrical variation in the excited nerve. Apparently the presence of special physiological factors must be assumed, whose effects are superposed on those of the purely physical factors. This point of