Protoplasmic Action and Nervous Action

The temperature-coefficient appears to be high in all cases. In frog's muscle and nerve Bazett and Adrian obtained Qio values of three or more.^ Burdon-Sanderson's observations on the frog's heart indicate values ranging from 2 to 2.5.^ The fact that the process of recovery shows this high coefficient seems to indicate its dependence upon processes of constructive metabolism: the temperature-coefficients are in fact similar to those of growth processes.

It is interesting to note that the various forms of protoplasmic transmission (in nerve, muscle, etc.), which theoretically depend on processes of breakdown, have usually shown temperature-coefficients of a dis- 4 Cf. Tait, loc. cit.; Trendelenburg, loc. cit.; de Boer, Journal of Physiology, XLIX (1915), 312; American Journal of Physiology, XL VII ^ Eckstein {Arch. ges. Physiol., CLXXXIII [1920], 40) finds a value of 2.6 for the refractory period of frogs' ventricle (average of 10 experiments between 5° and 20°).

tinctly lower order (Qio = 1.5-2).' The significance of this fact is not altogether clear, but it suggests that purelyphysical factors play more part in the destruction than in the reconstruction of the surface-film. There are general reasons for expecting that a process of structural breakdown, in which purely physical factors predominate, will have a low temperature coefficient; accordingly, since protoplasmic transmission is apparently dependent on such a breakdown, it is not surprising that its temperature-coefficient should be lower than that of the recovery process, which presumably is chiefly a result of metabolic reconstruction. Let us suppose that the first stage in the local excitation-process consists in a removal or destruction, by chemical reaction, of those constituents of the plasma membrane which are responsible for its semi-permeabflity and coherence; and that the second stage, immediately following, is some purely physical process of disintegration or falling apart, in which diffusion-processes are the chief factor. Then the chemical temperature-coefficient will be shown by the first stage only, while the second and probably longer stage wiU have the coefficient of diffusion-processes. The whole process will then have a low temperaturecoefficient similar to that observed, as the following simple calculation shows.

We assume that the total period of breakdown at 20° has a duration of 3 o- (about the duration of the rising phase of the bioelectric variation in frog's voluntary muscle), and that of this period one-third (i o") is occupied by the initial chemical decomposition (CaoO? with Qio = 3, and the remaining two-thirds (20-) by a physical disintegration (PaoOj with Qio=i.3 (the temperaturecoefficient of diffusion processes). Then: (i) Total duration of breakdown at 20°, C2o°+P2o° = 3 o") (2) total duration of breakdown at 10°, Cio°+Pio°=i crX3 + 2 cX

This value is similar to those obtained by a number of investigators (Maxwell, Lucas, WooUey, Snyder, and Harvey)' for the transmission-process in nerve and muscle. The recovery-process, on the other hand, which apparently occupies the greater part of the refractory period, presumably depends chiefly upon the reconstruction of the surface-film by metabolic synthesis, and accordingly exhibits the high temperature-coefficient characteristic of chemical processes.

The general theory of the refractory period is thus closely related to that of the stimulation-process as a whole. If stimulation is in fact dependent on an alternate breakdown and reconstruction of the surfacefilms of the irritable elements, we should expect irritability (which depends on the state of the film) to vary during the successive stages of the stimulation-process in a manner very similar to that which we observe. Evidently there are two distinct processes involved in the local change of stimulation, corresponding respectively to the ' ' absolute ' ' and the ' ' relative ' ' periods. The

view has long been entertained (by Hering and others) that the rising phase of the bioelectric variation is coincident with a period of breakdown of living material, and the return phase with its reconstruction.^ In Tait's experiments just described, the delay in the return phase of the variation was found to be associated with a delay in the recovery of normal irritability and conductivity; hence his proposal to identify the relative refractory period with the period of recovery or reconstruction of which the return bioelectric variation is the index. There are, however, various facts indicating that the return variation and the recovery of irritability may vary independently. Trendelenburg has shown that in cardiac muscle excitability does not return until some time after the bioelectric variation is completed; poisoning with muscarin lengthens the refractory period without greatly affecting the bioelectric variation.^ From general considerations a complete coincidence is hardly to be expected. Although the existence of a certain normal bioelectric potential between protoplasm and medium, the so-called '^ physiological polarization," is apparently necessary for stimulation, it is known that a tissue may be rendered inexcitable by conditions that have little effect on this potential, e.g., the presence of anaesthetics, Mg salts, etc. It is probable that the normal physiological polarization is regained rapidly during the early part of the refractory period, as shown by the completion of the return variation of potential; this change apparently indicates the return of the altered

surface-film to its previous continuous and semipermeable state; yet the film may require some further structural or chemical modification before it is in a condition favorable for stimulation and transmission. The relative refractory period seems to correspond to the time during which those regulatory or restorative changes are proceeding in the newly re-formed film. These general considerations receive support of an indirect kind from my recent experiments on transmission and recovery of transmissivity in passive iron wires immersed in nitric acid solution.^ In this inorganic model there is also a refractory or non-transmissive period immediately following the passage of an activationwave. During the progress of the chemical reaction following the activation of a passive wire in 60 per cent HNO3, the metal exhibits the chemical and electrical properties of ordinary or active iron, and is insusceptible to further activation. This initial period . of activity may be compared with the absolute refractory period in the Hving tissue. Its onset is accompanied by a variation of potential, the active metal becoming negative (anodal) relatively to its previous condition by ca. 0.7 volt. After a brief interval of vigorous chemical reaction, lasting from one to two seconds, the metal reverts spontaneously to the passive state, the effervescence ceases, and the potential immediately becomes again positive. During the first minute or so after this automatic repassivation it is found impossible to reactivate the metal completely by local mechanical or chemical treatment; e.g., touching the wire with zinc produces a brief and partial activation which is transmitted fo;*

only a short distance; by degrees transmissivity returns, and in one-and-a-half or two minutes (at 20°) is as complete as before. In this case the return of passivity — ^wdth the potential characteristic of that state — depends on the re-deposition of a thin surface-film of oxidationproduct. This process is itself a rapid one, as shown by the rapid change of potential from negative to positive; but the metal is at first relatively resistant to alteration, and regains its former properties only by degrees, probably as a result of a progressive thinning, rearrangement of molecules, or other change in the film, accompanying the approach to the equilibrium condition. If we may regard the processes in the inorganic model as resembling in their general features those of the irritable Hving system, it w^ould seem probable that in the latter the '' absolute '^ refractory period represents the early phase in the local stimulation-process during which the alteration and breakdown of the protoplasmic surfacefilm are in progress; while the "relative" period is that during which the film is being rebuilt and reconstituted in the succeeding recovery-process. As the film returns toward the normal or equilibrium condition, the ability of the tissue to respond and transmit excitation also returns. It is interesting to note that the temperature-coefficient of the process of recovery in passive iron is high and apparently similar to that of H\'ing tissues (Qio = 2-3).'

If during the local stimulation-process there is in fact a temporary breakdown or dissolution of the protoplasmic surface-film, a temporary increase of permeability to water-soluble diffusible substances should be associated with stimulation. The normal semipermeability of the Kving cell, implying impermeability to water-soluble substances of low molecular weight (neutral salts, sugars, and amino-acids) , depends on the structural continuity of the plasma membrane; and when this continuity is interrupted in any way, the effect is equivalent to a loss of semi-permeability. Such an effect may be temporary and difficult to detect in those cases where the surface-film is rapidly re-formed; but in the more favorable instances we should expect to find direct evidence of increased permeability during stimulation, in addition to the indirect indications afforded by the bioelectric variation and the refractory period.

According to the present theory, the local electric effects upon which the transmission essential to stimulation depends are the result of local alterations of the cell surface, involving increased permeability. Conversely, therefore, we should expect permeabihtyincreasing agents as a class to cause stimulation of irritable cells. The production of an "injury-current" by mechanical or other means shows that local interruption in the physical continuity of the cell surface forms a local circuit; and the formation of this circuit may be the means of starting a propagated disturbance or wave of excitation, in the same manner as a local interruption in the surface-film of the passive iron wire starts a wave of activation. It is well known that the application of a cytolytic, i.e., permeabihty-increasing, substance to a living tissue produces a local electrical negativity, giving rise to an injury-current, and that this current is

sufficiently strong to excite other irritable tissues. Hence the local production of such a current within the irritable element itself might be expected to cause stimulation in adjoining areas of the same element, an effect which would be repeated at the boundary of each area thus secondarily stimulated, and thus form the condition for a spread of excitation, in the manner already indicated. There is, in fact, a large body of evidence indicating that any rapid local increase of permeability, however induced, acts as excitant to an irritable cell or element. Mechanical treatment, like pricking or sudden pressure, the sudden application of heat, and rapidly acting cytolytic agents all have a stimulating effect on muscle or nerve. It is interesting to note that many cells, not ordinarily classed as irritable, undergo rapid and spontaneous structural alteration or breakdown under conditions involving local increase of permeability. This is well seen in the effects following the puncture with capillary needles of the surface of red blood corpuscles and other cells; frequently a disintegration starts at the point of injury and rapidly leads to the breakdown of the entire cell.^ In a red corpuscle thus treated the exit of haemoglobin may be seen to begin simultaneously over the whole surface.^ It is clear that in such cases the surface-film undergoes a sudden and irreversible increase of permeability; this change is propagated over the whole surface, and apparently in many cases through-

out the internal protoplasm as well, with the result that the cell breaks down. The rapid disintegrations characteristic of the ''explosive" blood corpuscles of Crustacea^ and of the vertebrate platelets, and the phenomena exhibited by other rapidly and irreversibly reacting cells like nematocysts and certain unicellular gland cells, appear to be examples of the same type of process. In all such cases the surface-film is stable only so long as its continuity is uninterrupted; interruption initiates a wave of disintegration which may involve the entire film-structure of the protoplasm.

In the cellular elements composing typical irritable tissues like muscle and nerve, in which the effects of stimulation are automatically reversed when stimulation ceases, the indications are that an essentially similar process of film-disintegration occurs during stimulation, but with the difference that a new surface-film is immediately re-formed. Whether the effects of stimulation are reversible or irreversible appears to depend on the ability of the protoplasmic system to re-form the structure necessary for stimulation. Under abnormal conditions, e.g., lack of oxygen, presence of depressant poisons, or disease, the rate of metabolic synthesis may be insufficient for complete restoration in the intervals of stimulation, and in such cases excessive stimulation may lead to the death of the cell. But, normally, recovery is rapid and complete in irritable elements of this class; as already pointed out, the indications are that the new film-structure is formed during the earlier part of the relative refractory period.

The ability of living protoplasm to form fresh semipermeable surface-films at cut or injured surfaces has long been known, and with the introduction of the methods of micro-dissection this property has lately become the subject of renewed investigation.' The rate and other features of the film-forming process vary widely in different forms of protoplasm, and also in the same form of protoplasm under different conditions; thus it is less active in cells that have been subjected to abnormal conditions than in normal and ''healthy" cells. ^ Apparently this process is of the same nature as the reconstructive change occurring at the surface of irritable elements after stimulation and occupying the first part of the relative refractory period. In many respects the refractory period resembles a brief period of fatigue; and it is well known that rapid recovery from fatigue, i.e., a complete restoration of the state preceding stimulation, requires that the supply of oxygen and the other environmental conditions should be normal. In the absence of these conditions excessive stimulation may lead rapidly to the physical disorganization and death of the protoplasm. Some of the effects of extreme fatigue (e.g., development of acid reaction, rapid onset of rigor) resemble those produced by cytolytic agents; and it is possible that certain metabolic products formed during activity have themselves a directly cytolytic

^ Cf, Chambers, American Journal oj Physiology, XLIII (191 7), i, for a description of film-formation after injury in egg cells. Also Jour. Gen. Physiol., V (1922), 189. 2 Seifriz {Annals of Botany, CXXXVIII [1921], 269) cites numerous observations on film-formation in protoplasm under various conditions; cf. also his article in Botanical Gazette, LXX (1920), 360. effect. Hence, their removal, independently of other conditions, is favorable to recovery. But the main factor in the reversal of the effects of stimulation, normal or abnormal, appears to be the synthetic or structureforming (formative) metabolism of the protoplasm. When the metabolic rate is rapid, recovery from stimulation or injury is prompt and complete, and vice versa. The difference between the recuperative and regenerative powers of young and old individuals in higher animals illustrates this condition.

Chemical or physical agents whose primary effect is to increase the permeability of the cell surface to watersoluble substances have as a class a strongly stimulating action on many irritable forms of protoplasm. The physiological effects produced by salts and combinations of salts in isotonic solution illustrate this very clearly. Thus pure solutions of neutral alkali salts, e.g., NaCl, have in general a rapid permeability-increasing action on living cells; this effect, if unreversed, is equivalent to toxic, and is prevented by the addition of a small proportion of CaCla or similarly acting salt to the solution; the antagonistic or '' an titoxic" action of the latter salt is to be referred chiefly to this preventive influence, which is equivalent to protective or anticytolytic. Correspondingly, pure solutions of Na salts cause stimulation or activation in many cells; and this effect also is prevented by addition of calcium in proportions similar to those required to prevent increase of permeability. The twitching of vertebrate skeletal muscle

in pure NaCl solutions has been well known since Sidney Ringer's time :^ this solution also causes rhythmical stimulation in motor nerve, although with a somewhat prolonged latent period;^ in both cases the effect is prevented by the presence of a little CaClz. The muscle cells of marine animals are also powerfully stimulated by pure isotonic NaCl; an especially instructive case is furnished by the larva of the annelid Arenicola, in which the contraction in the pure NaCl solution is very energetic. The body-cells of this organism contain a yellow pigment which serves as an indicator of increase of permeability. In the pure NaCl solution the pigment diffuses into the surroundings simultaneously with the contraction; and under a wide range of conditions the permeability-increasing and the stimulating effects of different salt solutions have been found to run closely parallel. Thus both effects are prevented or diminished by the addition of CaCU or MgCla to the pure Na-salt solution; and the addition of organic anaesthetics in the anaesthetizing proportions has a similar action.^ Similarly pure isotonic alkali salt solutions cause activation and increase of permeability in unfertilized starfish and sea-urchin eggs, and both effects are prevented by calcium salts or anaes the tics. "*

In general, any condition that prevents or retards the increase of permeability normally produced by the pure salt solution prevents or diminishes stimulation. The anti-stimulating or narcotic action of compounds like the organic anaesthetics or Mg salts appears to be associated with a characteristic action on the protoplasmic surface-films; the latter are rendered more stable, i.e., protected against the permeability-increasing or disintegrative action of the pure salt solution; as we have seen, a definite antitoxic influence is exerted by these compounds when they are added in appropriate concentrations to the pure salt solution. It is probable that this stabilizing influence is responsible for the general narcotizing properties of such compounds, since any condition preventing alteration of the surface-films must by that very action prevent stimulation, which is dependent upon such alteration.

The activating effect of pure alkali-salt solutions upon the unfertilized eggs of marine animals is a phenomenon which has many important physiological affinities with stimulation, and is exhibited under similar conditions. If starfish eggs are placed in pure isotonic solution of NaCl (or similar salt) for five to ten minutes (at 20°) and are then returned to sea water, a considerable proportion form fertilization-membranes and cleave, or even develop to a free-swimming stage. ^ But the same solution to which CaCla has been added (i mol. to 20 alkali salt) has little or no efTect.^ The eggs of Arbacia are more resistant to this type of salt action, and are only slightly affected by NaCl solution; but pure isotonic solutions of more energetically acting alkali salts, especially the iodides and thiocyanates of Na and K, form fertilization-membranes and initiate the activation-process in the same manner as other cytolytic

agents;^ in these eggs an after-treatment with hypertonic sea water is required to complete the activation. Solutions of these salts containing CaCla or MgCla are ineffective; pure isotonic solutions of CaCla and MgCla also fail to induce activation; apparently the initial effect of the activating agent must be to increase permeability, while the alkali earth salts have the reverse effect. This is probably the reason why when added to the pure alkali salt solution they counteract those effects (activation, stimulation, and toxic action) which are dependent on increase of permeability.

That the initial effect produced by the pure alkali salt solution is a permeability-increasing one is to be inferred from the general nature of the action of such solutions on living cells. The evidence is more direct in the case of pigmented eggs like those of Arbacia, in which the pigment visibly diffuses into the surrounding pure solution (of Nal, KCNS, etc.) after a few minutes' immersion. In the Ca-containing solution this effect is absent; the permeability-increasing and the activating effects are thus simultaneously prevented. A similar though less complete preventive effect is produced by various anaesthetizing compounds, especially higher alcohols; these when present in the anaesthetizing concentrations (i.e., those which arrest celldivision reversibly) retard or prevent the permeabilityincreasing and activating action of the pure solutions.^

Loeb's extensive researches on artificial partheno genesis have shown conclusively that permeabilicyincreasing substances in general (equivalent to cytolytic with long-continued action), whatever their special chemical nature, have the same activating effect on seaurchin eggs.' The substances used included numerous lipoid-solvent organic compounds, acids, bases, soaps, alkaloids, cytolytic glucosides (saponin, etc.), and foreign blood sera. A brief or superficial cytolytic action is thus regarded by Loeb as the initial or critical change in activation. This use of the term "cytolytic" seems, however, to be open to objection, since, as usually employed, it implies an irreversible or destructive effect on the cell; whereas the primary or critical change produced in activation is apparently a brief temporary increase of surface-permeability resulting from a rapidly reversed breakdown of the protoplasmic surface layer. The connection between this effect and activation is undoubtedly highly indirect and complex; but the same may be said for the connection between the direct action of any stimulating agent on an irritable tissue and the succeeding response of the latter. What is significant is that in both cases the primary or initiatory change in the complex physiological sequence appears to consist in a temporary disruption or breakdown (an effect probably related to de-emulsification) of the protoplasmic surface layer. This change furnishes the releasing condition for those metabolic and other processes of which the characteristic vital ''response" is the eventual and biologically important consequence or expression. Physical agents like ultra-violet radiation or heat (30-40°), which also cause cytolysis in unfertilized eggs

^ Artificial Parthenogenesis and Fertilization, University of Chicago Press (1913). and other cells, exhibit under appropriate conditions a similar activating influence.' Direct proof of the increased permeability of rapidly responding tissues like vertebrate muscle or nerve during normal stimulation is difficult to obtain. In the case of muscle the results gained with the method of electrical conductivity^ are of uncertain value, since the change in the form of the tissue, the production of electrolytes (e.g., lactic acid) in the process itself, and the change in the distribution or quantity of the intercellular fluids (lymph), all affect the total conductivity; hence the observed alterations may depend on other factors than the changing permeability of the plasma membranes. Direct observation of the penetration of easily detectable compounds into the muscle cell has given better results; recently Mitchell and his associates^ have shown that rubidium and caesium chlorides do not enter the muscle cells when the resting muscle is perfused with Ringer's solution containing these salts, but penetrate readily when the tissue is thrown into contraction by stimulating the nerve. In this case there seems to be an unequivocal demonstration of increased permeability to inorganic salts during stimulation. Embden

2 Cf. McClendon, American Journal of Physiology, XXIX (191 2), finds that frogs' muscle gives off inorganic phosphate to the medium during contraction but not during rest, and he regards this fact as further evidence of an increase of permeability during stimulation/ is immediately set up in the tissue, so that when the latter tissues to the electric current is largely or mainly a result alternating currents (which cause little or no polarization)

interpretation of this effect is that it is an expression of decreased polarizability. This view is confirmed by Bernstein's observation that the electro tonic currents of nerve, which are undoubtedly polarization currents, are also decreased during stimulation.^ Any such decrease of polarizability, in a system partitioned by membranes, is an indication of increased permeability of the membranes to ions. Recently Ebbecke has again shown that the polarizability of nerve is decreased during stimulation;^ he has also found that the same is true of the epidermal cells of the human skin, and he has discovered various interesting parallels between the phenomena exhibited by nerve and by skin, respectively, during and after the passage of the electric current.^ The polarizability of the skin and its resistance to constant currents are greatly decreased by either mechanical or electrical stimulation, and this effect is independent of variations of vascularity or other extracellular conditions. The reactions to the constant current are also typical: opposite effects are produced at anode and cathode, as in the polar stimulation of irritable tissues, and the action of salts and anaesthetics on the skin is analogous to that observed with other irritable tissues and cells; e.g., conductivity is increased by solutions of Na and K salts and decreased by Ca sa-lts and anaesthetics."* These

effects receive a consistent explanation on the theory of variations of permeability. The implication that apparently inert cells like epidermal cells are irritable, in the same sense as muscle and nerve, may seem a strange one, but it is in harmony with the general conception of irritability as an elementary property of all forms of living matter. Waller has pointed out that the most certain ''sign of life" in an apparently inert animal or plant tissue is the elicitation of an electric response (''blaze-current") on mechanical stimulation.^ The increased proliferative activity of the epidermal cells of the skin after hard mechanical usage is well known. Ebbecke's experiments show that such treatment increases the electrical conductivity; in other words, increases permeability and decreases polarizability, as in other cases of stimulation. The increased growth is the expression of this stimulation.

Recent observations by Crozier^ have also a bearing on the present problem. He finds that electrical stimulation causes- a well-marked increase in the permeability of the mantle-cells of nudibranchs to acids; in these cells the degree of permeability can be measured by the time required to change the color of an intracellular pigment, which acts as a natural indicator. Mg salts and anaesthetics (in appropriate concentrations) were found to decrease permeability, as in the cases already cited.^ Mechanical traction causes an increase of permeabihty, which within certain limits is reversible. These observations throw much light on the general

conditions of mechanical stimulation; related observations are those of Carlson, who observed that increasing the mechanical tension of the heart-ganglion of Limulus increased the rate of nervous discharge;^ and in frog's muscle it has been found that stretching causes an increase in the production of CO2 and lactic acid.^ Increase of permeability is probably a factor in both of these effects. The effects of mechanical, electrical, thermal, and other changes of physical condition on indifferent or unspecialized cells of various kind (blood corpuscles, egg-cells, epithelial cells, etc.) all have a bearing on the question of the relation of permeability-change to stimulation. The constant current causes polar disintegration in many cells, an effect analogous to polar stimulation ; similarly it causes polar secretion in cutaneous and mucous gland cells.^ The laking of blood corpuscles by induction shocks has also evident analogies to electrical stimulation.

There is abundant evidence of an increase in the permeability of the surface layer of many Qgg cells during the early stages of normal fertilization. In some cases the surface protoplasm undergoes extensive alteration and there results a visible loss or secretion of material to the exterior {Nereis, lamprey, frog).^ In the sea-urchin egg the space between the fertilization-membrane and the egg surface contains a colloidal substance which is apparently separated from the egg at fertilization.'^ Lyon has also observed an increased loss of catalase at this time.^ McClendon and Gray have shown that a significant increase in electrical conductivity occurs in the sea-urchin egg immediately after fertilization.^ Increase in the rate of entrance of dyes, and apparently also of toxic substances, has also been observed.^ In the Arbacia egg the rate of exchange of water in hypertonic or hypotonic media is increased several times as a result of fertilization.^ A change in the protoplasmic surface layer, associated with increased permeabiKty to w^ater and water-borne substances, thus appears to be a very general accompaniment of both normal and artificial activation. These facts, taken as a whole, suggest that the first stage of the activation-process consists in a breakdown, followed immediately by a re-formation, of the external protoplasmic layer or plasma membrane.