On the photochemistry of the retina and on visual purple

Front matter

THE LIBRARIES

I 1

COLUMBIA UNIVERSITY H

Preface

It is now more than twelvemonths since I undertook to publish an English translation of Prof. Kuhne's treatises on The Photochemistry of the Retina and on Visual Purple. Circumstances 'over w^hich I have had no control' have, until now, prevented my intention from being accomplished. Meanwhile further investigations have largely changed the aspects and the prospects of the subject. When Boll first announced his discovery of the sensitive purple colour of the retina he expressed the very natural conviction that it was directly connected with the act of vision; and, as will be seen from the following pages, the earlier results obtained by Kiihne promised at first to afford a facile solution of many visual problems. As, however, thanks to the energetic labours of this latter observer* and his talented assistant Dr A. Ewald, rapid pro-

1 Afl the reader is posHibly aware, Kiilane has in some quarters been reproached for having made use of Boll's discovery. To Boll must undoubtedly be given the credit of the discovery that the retinas of many animals possess ii Bensitive purple colour (for the mention of it by previous observers (see p. 15) does not amount to a discovery), and he therefore will rightly share in all the fame belonging to the subsequent developement of that discovery. At the same time the study of Boll's writings can leave no doubt on the mind of the candid reader that at first Boll did not realize that the visual purple uudorgoos

VI PEEFACE.

gress was made in our knowledge of the matter, it became step by step more and more evident that the mountain top was far more distant than had at first been imagined. From the first Kiihne seems to have felt, and he very early (see p. 18) stated his conviction that the visual purple was not 'the visual substance/ a knowledge of the changes of which would at once enable us to bridge over the gap between the waves of the luminous ether and the waves of the visual nervous impulses, but rather a type, shewing us, in a dim way, the manner in which the problems of the existence and behaviour of such a visual substance or such visual substances might be attacked; and, in the minds of the thoughtless, he may seem, in demonstrating (see Appendix, Note E) the possibility of vision and even colour vision without visual purple, to have destroyed, with his own hands, the value of his own handiwork.

More thoughtful consideration, however, will lead us to see reason to hope, rather than to despair, in such an apparent failure. The discovery of the visual purple, in the form in which it was first laid hold of by the general public, seemed, as people say, 'too good to be true ;' and it is no little consolation to know that the first premature delusions have been corrected with the least possible delay. On the other hand there remains the important fact that we can now point to positive photochemical

clianges through the action of light after the death of the animal; he attributed the bleaching after death to post mortem decomposition. It was Kiihne who was the first to observe that light bleaches the visual purple after death. This discovery made quite independently of Boll and in contradiction to Boll's statements is ample Justification for Kiihne's contiauing his investigations with a view to determining the bearings of what he 'himself had found out.' When the following pages have been read, it -will be at once seen that the bleaching of the retina by light after death is the key to the whole matter. In the second edition of my Text-Booh of Physiology (p. 414, 1. 19 from bottom small print, "which after a few seconds expostu-e to light," &c.) the desire to condense the greatest amount of information into as few lines as possible, has led me to use a form of expression which makes me seem to attribiite to Boll what really belongs to Kiihne : I gladly take this opportunity of correcting this inadvertence.

PREFACE. Vll

actions taking place in the retina : the first stone of a true theory of vision (i. e. of the origin of peripheral visual nervous impulses) has been laid. For some time past the minds of physiologists have been drawing near to the conception of a visual substance or of visual substances as the basis of vision. The speculations of Hering {Zur Lehre vom Lichtsinne. Wien. Sitzunffsherichte, LXVI. — LXX.), though in their present form they may be spoken of as crude, and as perhaps raising more difficulties than they remove, have this great merit, that by the hypothesis of a visual substance (or visual substances) they bring visual (and by analogy other specific) sensations into the same category as ordinary nervous impulses and even muscular contractions (we might perhaps go so far as to say as protoplasmic molecular movements in general), and thus open up the way by which the phenomena of a visual sensation, a nervous impulse, the act of secretion, and a muscular spasm may be made to illustrate each other.

Awaiting patiently the future discovery of the exact nature and relations of what may be called the true visual substances, we shall still be justified in maintaining that the visual purple is, if not directly at least indirectly, connected with vision; and, in this sense, I have not hesitated to retain the name ' visual purple,' in spite of the fact that frogs appear to see perfectly well in spite of the absence of any store of it in their retina.s, and that many animals which never at any time seem to possess such a store, have nevertheless very respectable vision ; and I trust that an account in the English language of its behaviour and properties will prove not unacceptable to the physiologists and psychologists of this country.

The translation has been made by Mrs Foster, who has found the task of converting Prof. Kuhne's somewhat idiomatic German into readable English not free from difficulty ; we tnist, howovor, that liis mcming has boon in all cases vm PREFACE.

correctly rendered, even if the diction should at times seem capable of improvement. I have gone carefully over the whole of the translation myself, and have added some notes in the form of an Appendix; and Prof. Kuhne has himself kindly revised the proofs.

M. FOSTER.

Part I. Photochemistey Of The Retina

F. I'

PART I.

PHOTOCHEMISTRY OF THE HETINA.

In a recent communication to the Berlin Academy, Herr Fr. Boll announced the beautiful and, beyond doubt, important discovery, that the bacillary layer of the retina of all animals is in the living condition not colourless, as has been hitherto supposed, but of a purple red colour. During life, says Boll, the proper colour of the retina is continually being destroyed by the light which falls into the eye; it is restored in the dark, and after death only remains a few moments. Animals which have been kept in the light are therefore very unsuitable for demonstrating the living colour of the retina, and the eyes of animals which have been exposed to the sun for a long time before death remain absolutely colourless. These facts illustrate the relation of the retinal colour to light on the one hand, and to vital conditions on the other.

Whoever has busied himself with the retina will be reminded by Boll's discovery (and thereby receive a wholesome admonition of the limits of his own ability), that he has already seen something of the kind before. He will perhaps remember that puzzling blood-clot — which at one moment he saw, or thought he saw, under the retina, and which the next moment disappeared. What he then passed over so lightly was nothing less than the key of the secret, how a nerve can be excited by light. In other words, it was the first fact disclosing the '•^istence of photo-chemical processes in the retina.

1—2

4 PHOTOCHEMISTRY OF THE RETINA. [PART

During my first attempts to test the truth of Boll's statements, I was under tlie impression, strengthened by what Boll had said, that the greatest haste was necessary in removing the eye, and taking out the retina; but I very soon convinced myself that a considerable time may be allowed for the operation, since the visual purple, as I call the proper colour of the retina, may continue to exist for some time after the retina has ceased to be in a physiologically fresh condition, and even - after death is only destroyed by light. Under the light of a good gas-lamp the retina may be quite leisurely spread out, and is then seen to lose its colour very gradually, for what takes place under bright daylight in half a minute, lasts in this case from 20 to SO minutes, that is to say, much longer than Boll gives as the duration of tissue-life. And in the dark, or in the light of a sodium flame, the purple does not pass away at all, either in the frog or rabbit, at least not for 24 or even 48 hours, in spite of decomposition evidently setting in.

By the discovery of these facts, the path of experimentation with the visual purple is freed from many difficulties. The necessary preparations can be conducted leisurely in a dark chamber lighted by a sodium flame, and the object then brought out for examination into diffuse daylight. A room such as is used by photographers, into which the light passes through yellow glass or paper, answers the same purpose, but less perfectly.

As w^e do not exactly know how long the rods or their parts remain alive after death, I treated the retinas of frogs in the sodium chamber with the most various reagents, many of which were without doubt capable of producing great structural alterations, to see whether the colouring and sensibility to light were thereby affected. The colour was destroyed by heating to lOO*' C, by alcohol, by glacial acetic acid, by soda solution, both concentrated, and 10 per cent. It was not affected by a solution of sodium chloride 0"5 per cent., by strong ammonia, by sodium carbonate solution, by saturated sodium chloride solution, by alum, lead acetate, acetic acid 2 per cent,, or tannic acid 2 per cent., by lying in glycerine for 24 hours, by ether, or

I.] PHOTOCHEMISTRY OF THE RETINA. 5

by being dried upon a glass plate. In all these latter instances, the retina, when brought into daylight, was found to be still red, and bleached more or less rapidly, the purple passing after 1 or 10 minutes into chamois colour\ which gradually disappeared, until there was scarcely anything of it visible. The depth of the colour naturally depended upon the other conditions of the retina, upon its being transparent, or cloudy. When it was opaque it gave an opportunity of proving the correctness of Boll's statement, that it is the outer layer, therefore really the layer belonging to the rods, which is coloured ; for an opaque retina is seen to be white in front, the red appearing at the back only. The colour comes out most beautifully after the action of ammonia, which makes the retina very transparent ; and the red of a retina treated with ammonia withstands the action of light from 10 to 20 times longer than an unchanged retina exposed to light of equal intensity. The dried membrane also retains its colour for a long time, but it also yields gradually to the light.

From what has already been said concerning the methods of obtaining coloured retinas, it is obvious that all kinds of liglit are not equally active in bleaching the visual purple. This is untouched by the photo-chemically inactive (or only slightly active) rays of the line i)^ It is only the very strongly coloured retinas (such as those of Rana temporaria) which shew any colour in sodium light, and then only a trace of it ; and sodium light is not quite monochromatic. The retina of a living rabbit examined with an ophthalmoscope in the approximately monochromatic sodium light, appears bluish white, with sometliing of a mother-of-pearl sheen, the vessels comingout with astonishing distinctness, like black lines drawn with ink. So also the pupil of an albino rabbit, when the eye is illuininuted obliquely, looks quite black. Indeed the sodium light, which is so easily rendered sufficiently intense, may be ' xpressly recommended as suitable for delicate ophthalmoscopic investigation.

In order to ascertain which are the particular rays which bleach the visual purple, I placed retinas spread out on glass ' .See Appendix, note A. ' Sec hmvcver p. r>7.

6 PHOTOCHEMISTRY OF THE RETINA. [PART

plates, in blackened moist chambers, covered them with glass slips, on which I had fastened strips of tin foil about one mm. broad, and then placed over them coloured plates of glass, or glass beakers filled with coloured solutions. For red I used blood solutions of such a strength that neither yellow nor orange were any longer visible in th^ir absorption spectra ; also red glass plates, which, however, allowed some amount of purple as well to pass through. For blue I used ammoniacal cuprie oxide, and for green, coloured glass plates, the spectrum of which consisted of only a narrow green band. Under the blood there was really no bleaching at all ; under the red glass the first trace of it appeared after 6 hours ; in the blue light the bleaching began after 2 hours, and in the green after from 4 to 5 hours. Of course, owing to the intensity of the light under which these experiments were conducted not being very great, and also not really comparable, one cannot from them arrive at accurate conclusions concerning the problem ; but the apparently more powerful action of the more refractive rays, particularly of the blue light, was brought out clearly \ On removing the cover-glass from the bleached preparations, there was seen, where the retina was protected by the strips of foil, a beautiful band of unchanged purple, in other words a positive photogram. I found the purple was as little changed by lithium light as by the blood-red, while, as might be expected, magnesium light very rapidly destroyed it. Once bleached, by whatever means, the purple never returned, either in the dark, or in different coloured light, or by being exposed to heat, or by the ultra red rays coming through a smoked glass illuminated by the sun^

After I had followed out the experiments I have described, with animals which had, according to Boll's directions, been kept in the dark, I was anxious to see how the retina of a frog would appear, which, immediately after exposure of the eye to light while the animal was still alive, had been prepared as rapidly as possible in a chamber of yellow light. According to Boll's opinion, I expected to see it bleached to a perceptible degree; I found it on the contrary as red as others. It is 1 See Appendix, note B. ^ See Appendix, note C.

I.] PHOTOCHEMISTRY OF THE RETINA. 7

therefore unnecessary to keep the animal in the dark just previous to the experiment. As the daylight was not very bright, the sky being cloudy, though quite sufficient for microscope work, I tried illumination with magnesium light, but that also was followed by the same result. I therefore came to the conclusion that Boll was mistaken in ascribing the failure that he once had in demonstrating the presence of the purple, to the fact that the frog had been kept in the light ; if he found his preparations equally bleached, this must have been due to exposure to light during the time they were being manipulated. To discover why it was that the visual purple remained thus apparently unchanged in the physiological act of vision, I isolated the retina of a frog and laid it on a glass plate, leaving the other in the bulb, which I had however extirpated and had opened wide by an incision along the equator. Both preparations were then brought into daylight which was still not very bright, and were left there until the first was perfectly bleached ; the second one was then brought back into a chamber lighted by sodium light, the retina taken out, laid upon glass, and brought out again into ordinary light ; it was at first of a deep red, but quickly became colourless. The sky not being clear, I conducted the same experiments with a magnesium lamp, and always found that the visual purple was restored so long as the retina remained in the eye lying upon the choroid, though it was protected from light and air by nothing more than thin capillary layers of the vitreous humour. On the following day, when the mid-day sun was almost clear, and shining so brightly that I was unable to look up at it, I repeated the experiment, by bisecting a frog's eye, removing the vitreous humour, and exposing the posterior half containing the retina to the sun for four minutes. I found even then red specks in the chamois-coloured retina, only the edges being completely bleached. In an entire bulb which I had exposed properly arranged to the same sunshine for 25 minutes, I still found very faint red spots in the midst of a great deal of chamois colour, but this was probably owing to the pupil becoming very contracted during the illumination. Since I always prepared the retina in sodium light in these cxperi-

8 PHOTOCHEMISTKY OF THE RETINA. [PART

ments, it might be thouglit that the period of photo-chemical rest thus occasioned, although short, in some way or other caused the return of the purple. This however is not the case, for if a divided eye be held up against the daylight for a period quite sufScient to bleach an isolated retina, and then, while the light is still falling on it, the retina be rapidly drawn out of the bulb, this will always be found to be at the first moment beautifully red. It is seen that my observations support those of Boll in this limited sense, that the retina may be bleached in living animals, after long continued exposure to direct sunlight ; I may moreover add that frogs which had been left to themselves for several days, in a glass case, in a very sunny place, at the end of that time had colourless retinas. While I do not agree with Boll's experience of the effects of "a moderately lighted chamber," we are at one as to the results of more intense illumination.

If the photo-chemical processes which take place in the retina separated from the eye, are taken as representing those which are going on in the living eye, the visual purple may be conceived of as continually being destroyed by the act of seeing, and by some process or other being as continually renewed ; and indeed Boll has expressed some such view as this. The oculist led by experience would immediately seek for the process of regeneration in the nourishment brought by the circulating blood ; for this is a favourite way of accounting for most of these kinds of events. In the case in point, however, the matter is far less complicated.

That which restores the visual purple is something nearer to hand, and cannot, in a frog's eye at all events, depend on the constant renewal of the blood, since an eye, when taken out and opened, exhibits the same apparent indifference to light as when connected with the whole body and the nutritive currents. If therefore we are correct in supposing the sensitive purple to be continually restored, the regeneration must proceed from something lying behind or on the rods, that is, from the retinal epithelium or from the choroid. Something must be there which either prevents the purple becoming lost, or re-creates

I.] PHOTOCHEMISTRY OF THE RETINA. 9

it. The idea at once suggested itself that the mere pigment as pigment had something to do with the matter, because a more intense action of light is to be expected if the retina, which usually only receives light from the front, is in addition lighted from behind, as is the case when it is spread out upon a white surface, instead of lying in its natural condition upon a velvety black ground. It could not however be supposed that this would protect- it so long and so completely as is found to be the case. Moreover, I could not discover that spreading out the retina with the rods turned downwards upon a dead black surface had much effect upon the time of bleaching ; and the following experiments will perhaps shew that the evident continual renewal of the sensitive colour is due to something else than the pigment, which, as is well known, is not present in albinos, and in many animals lies behind a tapetum.

As a proof that it is the choroid, including the retinal epithelium, which alone protects the purple from bleaching in light, it is sufficient to remove the retina in such a way, that a number of black shreds of the choroid still adhere to it, and then to spread it out on a thin glass slip, and expose it on every side. The manipulation for this purpose is not difficult, if the bulb is so cut out that a hole is left at the entrance of the optic nerve, and consequently the spot got rid of which offers resistance to the removal of the retinal membrane. From a bulb so manipulated, it is very easy to get a retina spread out without a crease, especially if a few meridian incisions arc made in it. These trivial matters are important, because in badly prepared and creased retinas the pigment actually prevents the access of light to certain parts of the retina. If now the dark strips are taken off the fully bleached preparation, the parts which lie under them will be found to be very deeply coloured.

Another experiment, which demonstrates the same tiling, consists in tearing the bisected bulb slightly until as usual some folds of the retina bulge out, then letting the light shine in, and quickly pulling out the whole retina. Where the folds were, wljite stripes will bo found, all the rest remaining red. 1 also made the following oxpcriincnt. Having made an

10 PHOTOCHEMISTRY OF THE RETINA. [PART

equatorial incision, I seized the retina along a considerable portion of its edge, and very carefully lifted it up from the pigment layer over a good half of its area. I then slipped a thin morsel of porcelain under the raised portion, and exposed the whole to daylight, until it was completely bleached. Of course the bleaching can only be ascertained with certainty in the flap which is raised, as the visual purple cannot be recognized in the dark reflecting hollow fundus of the eye. I next allowed the bleached retina to sink slowly back in sodium light on to its natural support, and to lie for some minutes upon it, making sure the meanwhile that my manipulation had been successful in avoiding the formation of creases ; and then I drew the entire retina away. It was uniformly red all over, and one could not even discover a line to mark the limit of the half which had been lifted up. A retina therefore bleached by light may regain its purple colour by simple contact with its natural back-ground. Still it remained to make the whole experiment in active light. This also succeeded, but the half which was restored was somewhat paler than the other. I have no doubt of the success of this operation undertaken by anyone, and I even go a step farther and recommend the cutting out of a flap, the bleaching of it upon a plate and the laying it back again upon the exposed pigment, by which it will be seen that any piece replaced in a normal manner always recovers its purple again. The regeneration has so often and so well succeeded with me by this method, that I was seriously led to try with a piece of tissue paper whether the cup of the eye did not contain a small quantity of red secretion; the morsel of tissue paper however came out moist it is true, but quite colourless.

In a frog's eye such experiments carried out with necessary care need not be hurried ; nevertheless the regeneration of the purple, unlike the mere continuance of the colour or the maintenance of its sensitiveness to light, is dependent on the action of living tissues, and when the tissues cease to be alive the regeneration can no longer take place. I have placed the eyes of frogs in 0'5 per cent. Na CI at 43" C. for 10 minutes, then bisected them, exposed them to the light, and always then

I.] PHOTOCHEMISTRY OF THE RETINA. 11

found the retinas colourless. Since the retinas of the eyes so heated were still red before exposure to light, they must have been bleached by the light. The same thing occurred in eyes which had died after being kept a day at about 20° C. It may be here mentioned that these failures in dead eyes prove that the pigment, considered as mere pigment and not as a living tissue, is ineffectual as regards the maintenance of the visual pui-ple.

If the regeneration of the purple depends on a continuance of vital activity in the pigmentary support of the rods, it is obvious that the rapidly decomposing organs of mammals are ill suited for these experiments. Here rapidity of manipulation is above all things necessary, and yet I have often succeeded in drawing off from the hinder half of the eye of a rabbit, beautifully red pieces of the retina, even after it had been exposed to the light for two minutes, that is for a time quite sufficient to bleach an isolated piece until the only colour left is that belonging to the blood-vessels. Still even in an albino rabbit, where the circumstances must be allowed to be peculiarly favourable, I think I have been able to distinguish between the colouring of a piece of retina which has remained in its natural position, from that of one which had been removed, especially when the former, after the bleaching of the latter, had been spread in the same way on porcelain. Nevertheless I cannot speak with certainty on this point, since the retinas of the specimens of albinos at my command for this demonstration were taken from a variety which just now is very difficult to get here, and in spite of being kept long in the dark failed to shew a really good intense purple, and after being exposed to light exhibited only a slight variable pale orange colour, which indeed is not an altogether unknown feature in mammalian retinas.

It would be especially interesting to investigate this orange

colour*, whicli perhaps co-exists in company witli the purple,

ince Boll has made the very important observation, that blue

rods are very frequently present in the retina of the frog.

That there are many albino eyes possessing a well-developed

' See Appendix, note B.

12 PHOTOCHEMISTKY OF THE EETINA. [PART I.

purple I saw in later experiments, of which I will give an account on some future occasion.

The last-named series of experiments lead me back to ihe conclusion that the maintenance after death of the vital conditions in the outer layer of the visual apparatus is to be recognized, not by the existence of the visual purple, and its destruction by light, but by its relative indifference towards light, and I think that the being able to detect this reaction in the frog's eye for so long a time after death shews a happy agreement with Holmgren's and McKendrick and Dewar's beautiful experiments upon tjie galvanic currents of the retina, and their changes during stimulation by light.

What part of the choroid serves to regenerate the purple can for the present only be conjectured ^ ; probably it is not to be sought so much in the dermic layer of the choroid as in the epithelium in which the rods are embedded, and which has been rightly considered as part of the retina. The retina, so long as it is maintained in its natural connections with this epithelium, resembles not so much a photographic plate as a whole photographic workshop, in which the operator, by bringing new sensitive material, is always renewing the plates, and at the same time washing out the old image,

^ See Part 11. and Appendis, notes D and F.

Part II. Visual Purple

PART II.

VISUAL PURPLE.

The conjecture which was expressed in the introduction to the former treatise, that the red colour of the retina had been seen by many observers long before it had obtained so great an interest through Boll's communications, may be readily confirmed by studying the rich literature on the structure of the retina. Apart from A. Krohn's discovery of the red rods in the Cephalopoda quoted by Boll, their occurrence in vertebrates was first suspected by H. Muller in 1851. The importance of the subject will justify a verbatim quotation of the essential statements.

A. Krohn says in his paper delivered on Sept. 4th, 1839*, " Close by the dark stripes the fibres (visual rods) are of a reddish colour, but at their ends turned towards the vitreous membrane quite colourless ; the transparency and the rose-red sheen of the inner surface of the retina are thus accounted for."

This is confirmed by Hen sen in p. 39 of his paper on the eyes of Cephalopoda'' in the following words : " In the fresh retina they (the rods) possess red, shining, homogeneous contents."