Plant Response as a Means of Physiological Investigation

Possibility of two distinct stages of reversal, A and B. Further, since the effects at anode and kathode are, generally speaking, contrary in character, we might expect a corresponding change, but of opposite nature, to make its appearance progressively at the anode. In other words, it might happen that at a certain stage in the raising of the E.M.F. the exciting value of the kathode would be considerably diminished, and that the anode would begin to show excitatory effect. This might be designated as the A stage.

On still further raising the E.M.F. the same contrarydirectioned change might be expected to continue progressively at the anode and kathode, and to reach a stage at and beyond which it would be the anode which excited at make, while the kathode produced either no excitation or actual depression. This might be designated as the B stage. We should then have a complete reversal of the normal polar effects. If we exhibit these inferences, as to the relative excitatory powers of anode and kathode with increasing E.M.F., by means of curves, whose abscissae represent the E.M.F. while their ordinates give the corresponding excitatory values, the kathode curve would first rise to a maximum, and then fall continuously, till, reaching the zero line, it might even proceed still further in the negative direction, thus representing depression. The anodic curve, on the contrary, would at first descend in the negative direction, thus indicating increasing depression of excitability, until it reached a negative maximum, after which there would be a reversal, and it would begin to ascend and reach the zero-line. Here the anode would cease to depress. After this it would proceed upwards in the positive direction, indicating a continuously increasing power of excitation. The anodic and kathodic curves in the course of this ascent would cross at a

certain point, indicating that both of them now excited in about equal degrees. This would constitute the A stage. Beyond this would be the B stage, where the anode alone would excite. I now proceeded to subject these theoretical inferences to the experimental test. The special difficulty of this investigation lies in the fact that it is necessary to discriminate the direct from the transmitted effect at the two electrodes, with absolute certainty. For if the two points be not at a sufficient distance, and if the conductivity of the intervening tissue be great, the true effect of one electrode may be rapidly transmitted, and appear at the other. For these reasons, the plant Biophytum is not in this case a very suitable subject for experiment, its conductivity being great, and its opposite leaves not at a sufficient distance from each other. Mimosa, however, may be made to serve the purpose, for, though its conductivity is great, it is possible to select two leaves on different branches of the same plant which are very far apart. The plant Averrhoa is also appropriate, its conducting power being relatively slight. Electrical connections may, in this case, also be made with opposite leaves widely apart. In the case of Mimosa the excitatory effect is made visible by the fall of the leaf, in the case of Averrhoa by the depression of the leaflets. It is thus possible to render the effect of the two electrodes mutually distinct. It is also possible to distinguish the transmitted excitation, if any, by the serial depression of the intervening leaves or leaflets during the passage of the wave of excitation.

As these excitatory effects are dependent on the physiological condition of the tissue, we should expect that the E.M.F. which produces reversal would vary with different plants and their physiological conditions. The highest constant E.M.F. available for my own investigations was 220 volts, that being the pressure in the street-mains. I therefore hoped that I might be fortunate enough to find plants in a state to exhibit the expected reversal within this value. I shall now proceed to describe actual experiments with various

plants, and first I shall take those in which the A stage was exhibited, that is to say, those in which the anode as well as the kathode showed excitation at make. Experimental verification of A stage effects. — With the plant Biophytum, I have always found, without exception, that up to thirty-two volts, or thereabouts, the polar effect was normal ; that is to say, excitation was produced at the kathode at make and not at the anode. On using an E.M.F. of fortyeight volts, however, with a certain specimen, I obtained excitatory response at make, at both anode and kathode. That this anodic effect was not due to transmission of excitation from the kathode, was seen in the fact that some of the interpolar leaflets were not affected, as all would have been had the wave of excitation passed from kathode to anode.

I shall next describe experiments made on Mimosa, in which, as has been said, the two electrodes can be separated by a longer tract of tissue. In the case of this plant, the value of the E.M.F. which is required to bring on the A stage effect, is much higher than in Biophytum. I have occasionally obtained it with no, but more usually with 220 volts. In order to show how at this stage the anodic and kathodic effects tend to become interchangeable, 1 shall describe three experiments.

In the first of these, an E.M.F. of 1 10 volts was used. At make, the kathodic leaflets fell energetically, while the anodic fell but slightly, and after a little delay. Here we see that though reversal is setting in, yet the normal kathodic effect is relatively predominant. In the second of these experiments, I used 220 volts. The anodic fall now took place slightly earlier than the kathodic. The current was maintained till the leaflets recovered. On now breaking the circuit, there was a slight anode-break excitation, but none at the kathode. In this case, though from the slight priority of the anodic excitation we infer some predominance of the anode, yet the fact that the effect at break is normal shows that we are still in the

transition stage. In the next experiment, the tendency to reversal will be shown to have become predominant. In this third experiment with another specimen, an E.M.F. of 220 volts was again used. At make, there was an immediate energetic fall of the anodic leaflet, while that at the kathode was slight, and delayed for some time. At break, moreover, there was no effect on the anode, and a slight and delayed excitatory effect was distinctly perceptible at the kathode. From this we see that the anode is now appropriating the normal action of the kathode, and vice versa, reversal having set in unmistakably.

Reversed action in protozoa.— These experiments will probably be found to explain the assumed anomaly in the case of protozoa. In experimenting on Actinosphczrium, for example, Verworn found that ' at closure of the current in the first place, the pseudopodia, both on the anodic and kathodic side of the globular body, become varicose and begin to contract. If the circuit be opened the pseudopods on the kathodic side become varicose in about the same degree as had taken place immediately after the closure of the circuit' 1 In this experiment, where both anode and kathode exhibit excitation at make and only the kathode at break, we have a case exactly parallel to that of the third experiment with Mimosa, which has just been described. That, as in the case of plant-tissues, a fairly high E.M.F. was instrumental in producing reversal appears probable, from the fact that it is specially mentioned in the account of the experiment, that ' in consequence of the high resistance in the circuit, a comparatively high E.M.F. had to be used.'

Experimental verification of B stage effect. — I could not obtain with Mimosa at 220 volts complete cessation of excitation at the kathode at make, but I succeeded in doing so with Averrhoa, in the autumn and winter seasons. With this plant, I observed all these A stage effects, which have already been described in the case of Mimosa ; and that of completed reversal, or the B stage effect, was obtained in more than a dozen instances, out of which I shall give an account of two.

In the first of these, the two electric contacts were made on the same leaf, at a distance of 5 cm. from each other. At make, using an E.M.F. of 220 volts, excitation was produced at the anode only, and the depression of successive leaflets proceeded towards the kathode, but was arrested at one pair in advance of that point, the kathode apparently acting here as a depressor. In the second experiment, the electric contacts were made at a great distance from each other, with middle points of two opposite leaves. At make, excitation was produced at the anode only. At break, however, it took place at the kathode and not at the anode. We have here a complete reversal of the normal polar effects under the action of very high

The following tabular statements show at a glance the polar effects at both A and B stages, under a high E.M.F. : Law of polar effects under high E.M.F. — We have now traced out that process of continuous change by which under a gradually increasing E.M.F. there is produced a reversal of normal polar effects, and we thus arrive at the following law of polar excitation : Under high E.M.F. — at the A stage — both anode and kathode excite at make; at break there is occasional excitation at either anode or kathode. Under excessively high E.M.F. — at the B stage— the anode excites at make and the kathode at break.

Without this addition the law of polar excitation is incomplete ; and I shall have occasion, in my work on the Electro-Physiology of Plants, to show its application in explaining certain excitatory electromotive phenomena which would otherwise have remained obscure. Investigation on polar effects by death-response. — I have already explained in Chapter XV. that the deathpoint of an excited tissue is lowered below the normal. This made it possible to devise a test by whose means it might be determined which of the two electrodes produced excitation. Thus, on taking two similar petals of Passiflora, and making one anode and the other kathode, it was found with a moderate E.M.F. that death-discoloration took place at the kathode, at a temperature of 40 C. lower than at the anode (p. 185), thus proving that under these conditions it was the kathode which produced excitation. I was now desirous of finding out whether the same test could not be applied to the demonstration of the reversed effect due to high E.M.F., and in this connection I shall give an account of an experiment on the coloured petals of Sesbania coccineum, the deathdiscoloration of which occurs normally at 65*5° C. Taking two similar petals, and using the high E.M.F. of 220 volts, I found on sending a current that death-discoloration took place at the anode, at 6o° C, that is at 6\° below the normal. The discoloration point of the kathode was also lowered, but only slightly, being 2\° below the normal. We thus see that with a high E.M.F'. it is the anode which is more excitable at make. It is clear from this that the reversal of the normal polar effect has set in, the anodic excitation being considerably predominant.

Reversal of polar effects as due to fatigue or tissuemodification. — It has now been demonstrated that an excessively strong E.M.F. is one of the conditions by which the reversal of normal polar effects may be brought about. We shall next study other circumstances which may also be efficient to induce this reversal. This subject assumes the greater importance from the difference of opinion which exists among investigators in animal physiology as to the possibility of such reversal. The question has not yet, as far as I am aware, been definitely settled. Thus, ' Aeby thought he had proved that under certain conditions, more particularly with progressive fatigue of the preparation, the normal reaction — in which the excitatory action of the kathode far exceeds that of the anode — -was exactly reversed. Aeby's experiments, however, are by no means unimpeachable, as both Engelmann and Hering pointed out later. Engelmann, also, came to the conclusion later, that such a complete reversal of phenomena {i.e. of the law of polar excitation) might take place. But until it has been determined by unexceptional experiments, there must be great scepticism in regard to such statements.' x

We now turn our attention to that of the changed condition of the tissue by which the normal polar response may become reversed, and in this regard the experiments which I shall describe are very instructive, as these changes are there seen to occur progressively. I took a specimen of Mimosa and carried out on it five consecutive experiments. The two electrodes were attached to the pulvini of different leaves on the same stem, and the E.M.F. used was fifty volts ; an interval of about seven minutes was allowed in each case for recovery. For easy inspection, the results are given in somewhat tabular form.

(i) At make— Leaves fell both at kathode and anode. The kathodic fall was earlier and more energetic. (2) At make — The kathodic leaf fell, and the anodic fall was slight. at anode. The action of anode-break was here much stronger than that of kathode-make. (3) At make— Fall of kathodic leaf; no action at anode. At break — No action at kathode ; response at anode. (4) At make — Kathodic action became feeble, and anodefall, though at make, the more pronounced of the two.

(5) At make. — No action at kathode; feeble action at anode. In tracing out the changes which are here taking place at each electrode, we are struck by their progressive character. If we fix our attention first on the kathode, we find that the normal effect in the first of the series is gradually diminished, till it disappears in the last. Again taking the anode, we find a still more remarkable change, of a periodic character. In the first experiment, we observe the most pronounced abnormality, or reversal of the series, inasmuch as there was response at make and none at break. In the second, the response is tending towards normal, the anode-make effect being feeble, and the break strong. In the third, the anodic response has become normal, for there is no action at make, but excitation at break. In the fourth, we again see a tendency towards reversal, inasmuch as again there is response at make and none at break. The same state of things, though in a less degree, occurs in the fifth experiment.

We have thus observed two different conditions, each of which may contribute to produce this reversal of polar effects. These are, firstly, the influence of a high E.M.F., which, at or beyond a certain critical value, will produce reversal ; and, secondly, certain tissue-modifications similar to those which we have observed during the progress of fatigue. It is clear that with slight tissue-modification the critical value of the E.M.F. at which, under normal conditions, reversal of polar effects would take place, will be lowered. This, the experiments on Mimosa just described clearly show ; for in them we see that reversal has set in at the relatively low E.M.F. of

fifty volts, whereas normally in Mimosa the critical value is considerably above a hundred volts. These tissue-modifications sometimes proceed so far that I have occasionally observed reversal in the case of this plant even with a moderate I was next desirous of determining whether these different types of polar effects — normal, transitional, and reversed — could not be demonstrated in some novel and striking manner, in the case of animal tissues. It occurred to me that the intermittent flashes of light emitted by the firefly might be simple expressions of rhythmic excitation, a subject which will be dealt with in detail in Chapter XXI I J. The emission of light, or an increased intensity of emission on the part of the insect, would in that case be indicative of the state of excitation, and this mode of excitatory expression I shall designate as glow-response.

Investigation of polar excitation by glow-response — I may here state in anticipation that I have succeeded in demonstrating, by means of this glow-response, all the principal characteristic effects of (a) normal response, due to moderate electromotive force ; (b) the reversed effect due to high electromotive force ; and (c) the reversed effect due to a modified condition of the tissue. It may be pointed out further, that some specimens gave the normal, and others, owing to a modified condition of the tissue, the reversed effect ; but that the results obtained from any given individual were always consistent and characteristic.

I shall first describe certain results which were frequently observed, and which are entirely analogous to those described in a previous chapter as given by a nerve-and-muscle preparation, and highly excitable tissue of Mimosa (p. 197). We there saw that while the current was ascending, the exciration exhibited by the terminal organ at make was due to direct action of the proximal kathode. Excitation was also produced at break, and this was due to the transmission of the distal anode-break effect. Again, when the current was reversed, excitation was exhibited in a corresponding manner,

through the action of the distal kathode make and the proximal anode-break. The firefly under natural conditions emits flashes of light at intervals of about three seconds, from two discs, situated on the ventral surface of its tail. We select a specimen and make suitable electrical connections, one with the head, and the other with the luminous disc. The natural luminescence of the insect is moderate and intermittent ; but on now passing through it a descending current from a battery having an E.M.F. of twelve volts, the light at once becomes persistent and very brilliant. We must bear in mind that the luminous discs stand here in the place of the terminal motile indicator, of the nerve-andmuscle or Mimosa preparation, and that the state of excitation is indicated in them by the increase of luminescence instead of by an excitatory movement. This glow-response, then, is due to the action of the proximal kathode-make. The induced brilliance slowly dies down, and in the course of a minute and a half becomes very feeble. If the circuit be now broken, a single intense flash is produced, due to the excitation of the distal anode-break. The insect now recovers from the state of induced excitation, and begins once more to exhibit its natural intermittent flashes. We next pass the current in the reverse, that is to say ascending, direction. The light again becomes persistent and brilliant, owing to the excitatory action of the distal kathode-make. During the continuation of the current, the light wanes and becomes feeble. But when the circuit is broken, there is once more seen a single flash of intense light, due to the action of the proximal anode-break.

In order to ensure a simpler condition for experiment by eliminating the nervous conduction of excitation, I next isolated the double disc, and found that the detached organ maintained its excitability for a couple of hours or more. The discs now emitted a light which was somewhat feeble but not intermittent. Electrical connections were then made with the two discs, by means of fine cotton threads, moistened with saline solution, and an E.M.F. of sixteen volts was used.

At make the kathodic disc was found to become very brilliant, and there was no effect on the anodic. In some instances, indeed, the anode became dimmer than usual, thus showing the depressing influence of the anode. At other times, again, the luminous excitation of the kathode irradiated and encroached upon the anodic region. At break it was the anode which flashed out, showing excitation. These results, as will be seen, are entirely normal. I shall next describe experiments which illustrate the reversed effect sometimes observed with excessively high E.M.F., and at other times due to a modified condition of the tissue.

With regard to the production of the reversed effect under a high E.M.F., some difficulty is encountered owing to the proximity of the two discs of the luminous organ. The effect of one electrode is thus liable to encroach on the region of the other. But specimens are occasionally obtained in which, the conducting power of the tissue being feeble, each effect is practically confined to its own area, though the excitatory E.M.F. may be high. In the following investigation it is to be noted that successive experiments were carried out on the same specimen, without disturbing the electrodes. By proper manipulation of the key, the current was made to flow now in one direction, then in another, or the acting E.M.F. was changed from low to high at will. The differences of the results observed must therefore have been due, either in the first case to the reversal of anode and kathode, or in the second case to the difference in intensity of the E.M.F.

A specimen was taken of the detached luminous organ, and electrical connections were made with the two discs, by means of moistened threads. An E.M.F. of ten volts was first used, and the effect at make was a brilliant illumination of the kathode-disc. During the continuation of the current this gradually waned, but at the break of the circuit a brilliant flash appeared at the anode. Thus we have, in the present case, the normal effect with moderate E.M.F. I next used with the same specimen the high E.M.F. of fifty volts.

The luminescence at make now took place at the anode, and at break at the kathode. On reversing the current, the new anode, formerly kathode, gave responsive illumination, and at break the new kathode responded. In these results, therefore, it will be seen that we have an instance of reversal of polar effects, under excessively high E.M.F. These reversed effects are usually observed with a high E.M.F. ; but sometimes, as has been said, owing to a modified condition of the tissue, they may be obtained, under the action of even a moderate E.M.F. I shall now give a very interesting example in which we can trace the process of reversal owing to the modification induced by fatigue, in a manner somewhat similar to the last experiment described in the case of Mimosa (p. 209).

I took a fresh specimen of the detached organ, and carried out four successive experiments on it, observing the effects at both make and break, the E.M.F. used being twenty volts. In order to present these results at a glance, I shall again put them in a somewhat tabular form. (1) At make — Luminous response at kathode, which irradiates slightly towards anode. At break — Little effect at anode, but natural luminosity of the kathode falls below par. This shows the depressing action of kathode-break.

At break — Luminous response at kathode only. These effects, especially that of break-excitation at kathode, show that the condition of reversal has set in. This will become still more pronounced in the succeeding experiments. (3) At make — Luminous response appears at anode and irradiates slowly towards kathode. (It will be seen that we have here a complete reversal of the effects observed in (1) at make.) At break — No immediate effect is at first observed ; later, a flash passes from anode to kathode.

At break— No effect at anode, but feeble augmentation of luminosity at kathode. These results afford us some insight into that obscure phenomenon of the modified condition of tissue by which reversal of response is brought about. We have seen that in the case of Biophytum, the polar effects are always found to be normal, within rather a wide range of E.M.F., that is to say, up to about thirty volts. The kathode here excites at make, and the anode at break. I have carried out several hundreds of experiments with this plant, but have not once come across any deviation from this normal action.