Abstract
CONTRIBUTIONS FROM THID ZOOLOQICAL LABORATORY OF THID MUBIDUM OF COMPARATIVE ZOOLOQY AT HARVARD COLLEQE. NO. m THE MOVEMENTS IN THE VISUAL CELLS AND RETINAL PIGMENT OF THE LOWER VERTEBRATES LESLIE B. AREY Northwestern University Medical School ONE TEXT FIQURE AND FIVE PLATES CONTENTS 1. Introduction and historical review. ..................................... 121 2. Material and technique.. ............................................... 124 ........ 126 ........... 127 (b) Visual cells.. ................................................. 175 E. Effect of oxygen.. F. Interrelation of in 4. Discussion.. ........................................................... 181 ...................... 184 ... , ..... ...... 188 1. INTRODUCTION AND HISTORICAL REVIEW The positional changes that occur in the vertebrate retina chiefly through the action of light have attracted the attention of many workers in continental Europe although, strangely enough, neither English nor American investigators have hither- to concerned themselves with this particular field of endeavor. The results acquired from these researches have proved of such interest and significance as to equal, and perhaps even to sur- THB JOURNAL OF COMPAIUTIVID N~UROLOQY, VOL. 26, NO. 2 APRIL, 1916 122 LESLIE B. ARET pass, those obtained from the two other branches of retinal physiol- ogy, namely, the study of the visual purple and of t,he action currents in the optic nerve. In order that the reader may understand the present status of &is subject, it is desirable to summarize briefly the general conclusions that have been established relative to the efficiency of light in producing movements in the retinal elements. For a more extensive review of this literature reference may be made to a separate paper by the writer (Arey, '15) or to the excellent compilation of the German author, Garten ('07). Although a variability in the position of the retinal pigment had been noted by early workers (H. Miiller, '56; Morano, '72) the cause remained unsuspected until Boll ('78) and Kiihne ('77) independently discovered that in the light the retinal pig- ment of the frog extends nearly to the external limiting mem- brane whereas in darkness it retreats, thereby forming a com- pact layer next to the choroidl (figs. 1 and 4). Later obser- vations have corroborated Kuhne's original view that pigment migration is not due to the extension and retraction of cell proc- esses but to the movement of pigment granules in the proto- plasm of relatively fixed cells. The most extensive pigment migration occurs in fishes (Stort '86) and in anuran amphibians (Boll, '78, and Kiihne '77, on the frog; Arcoleo, '90, on the toad), whereas the positional changes in the pigment of urodeles are relatively limited (Angelucci, '78). Well defined movements of the retinal pigment are also found among birds, including not only those that are diurnal, but also some that are nocturnal in habit. Among reptiles (Angelucci, '90) and mammals (Angelucci, '78) limited pigment changes have probably been detected, notwithstanding the contradictory evidence presented by various workers. Czerny ('67) found that when sunlight was concentrated by a lens on the retinas of various animals, the pigment became more highly expanded in the affected regions than in other portions which had been exposed to light but not so treated. In his experiments no comparisons were made with dark-adapted retinas. These somewhat pathological tests were not well substantiated by the later work of Deutschmann ('82). MOVEMENTS IN THE VISUAL CELLS 123 Movements of the inner member of the vertebrate cone cell were first observed by Stort in 1884, although the earliest an- nouncement of this discovery was published by Englemann (’85) in whose laboratory Stort worked. To the contractile portion of the cone’s inner member Englemann applied the sig- nificant term ‘myoid’ (figs. 30, 31; my. con.). The contractility of the myoid is extraordinary, since in some fishes light produces a shortening of this part to 10 per cent of the length which it assumes in darkness (figs. 25, 27). If effective at all, light always causes a shortening and darkness an elongation of the cone cell. Stort (’87) extended his first discovery on the frog by experi- mentation upon representatives of the various other vertebrate classes, thereby showing that in fishes and birds extensive move- ments of the cones likewise occur. In the salamander, as a type of urodele amphibian, responses of the cones to light have both been asserted (Angelucci, ’go), and denied (Garten, ’07). Among a few reptiles (Englemann, ’85) and mammals (Garten, ’07) slight changes have apparently been detected. The visible response of the rod’s inner member presumably is not identical throughout all the vertebrate classes. Angelucci (’84) was the fist to observe a shortening of the frog’s rod after exposure to light and later, in 1890, he applied the term ‘myoid’ to the contractile portion of the rod’s inner member (figs. 30, 31; my. bac.) in a sense similar to that in which Englemann has previously used it for the corresponding portion of the cone; Arcoleo (’90) likewise reported that the rod myoid of the toad shortens in the light. Recently, however, Lederer (’08) has asserted that the photomechanical change in the rod myoid of the frog is not a shortening but an elongation. In all fishes that possess cones, the rod myoid lengthens in the light and shortens in darkness, as Stort (’87) first believed. The response in the rods of day-birds (Stort, ’86) is similar to that in fishes, although in night-birds (Garten, ’07) movements of these cells may be entirely absent. No experimentation has been per- formed upon the rod cells of mammals or of reptiles; in the latter group, however, the retina usually lacks these elements. 124 LESLIE B. AREY 2. MATERIAL AND TECHNIQUE The work of Chiarini ('04a, 'OS), in which he systematically investigated the effect of light on the retinas of representative vertebrates, led him to the conclusion that the maximal changes in the position of the pigment and of the cone cells occur in the lower vertebrates and particularly in the fishes, whereas in the highest vertebrates these changes are extremely limited or may even be imperceptible. For experimentation involving varia- ble temperature poikilothermous animals are necessary, and no vertebrates are more easily available for this purpose than are is added the ad- fishes and amphibians. When to availability vantages which they offer by the possession of exceptionally well developed visual and pigment cells, which are capable of undergoing the greatest dimensional changes found in any of the vertebrates, it will be seen that these animals are particularly favorable for such physiological studies as were conducted in the present investigation. The following fishes were used: the common horned pout (Ameiurus nebulosus Lesueur) ; the common killifish (Fundulus heteroclitus Lim.) ; the shiner (Abramis crysoleucas Mitchill) ; and the goldfish (Carassius auratus Linn.). Of these, all but Fundulus are fresh water forms, while Fundulus is found in brackish water, and especially at the mouths of fresh water streams. Abramis and Carassius belong to the same family, the others to different families. Goldfish were obtained for the most part from dealers although a number of feral animals were used. Many of the streams and ponds about Cambridge are stocked with feral goldfish, liberated by accident or design. The majority of such animals have lost their gold coloration and have returned almost completely to the original olivaceous type- The amphibians used were as follows: the leopard frog (Rana pipiens Schreber) ; the green frog (Rana clamitans Latreille) ; the bull frog (Rana catesbiana Shaw); and the mud puppy (Necturus maculatus Raf.). Of the three species of frogs, the leopard frog was used almost exclusively for experimentation on adult animals; the larvae only of the bull frog were employed. MOVEMENTS IN THE VISUAL CELLS The technique of preparing retinas for microscopical examina tion was very simple. Eyes were removed from their orbits in two ways. In Ameiurus, where the eyes are prominent and the skin is soft, excision was performed directly. The eyes of amphibians and of fishes other than Ameiurus, particularly in experiments conducted in the dark where rapidity of operation was desirable, were not excised directly but according to the following procedure. With heavy scissors the cranium was bisected in a median sagittal plane; following this, a trans- verse cut just posterior to the orbit freed the two halves of the cranium, with the contained eyes, from the rest of the body. In either case the operation was performed in a few seconds and the eye, without being handled, was allowed to drop into the fixing fluid.2 Both Perenyi's and Kleinenberg's fluids gave good fixation (Howard, '08; Palmer, '12), but of the two, Perenyi was pre- ferred. The toughness of the sclera and the consequent slow- ness in the penetration of fluids demands generous allowances of time during the various steps preparatory to imbedding in paraffine. Dehydration and clearing in xylol should, however, progress as rapidly as possible since otherwise the sclera becomes extremely hard. Two methods were used in removing the lens, one of which, although longer, gave much more satisfactory results. The first somewhat tedious procedure consisted in paring away the front a razor after the eye had been previously face of the eyeball with imbedded in paraffine. After removing the face of the eyeball slightly beyond the ora serrata, the lens was pried from its paraf- fine matrix with a dissecting needle; following such manipula- tion reimbedding was of course necessary. The second and simpler method was to remove the face of the eyeball with fine curved scissors after the eye had been hardened in absolute alco- hol; if, however, the eye was not sufficiently hardened or the 2 A method of simple decapitation used by Pergens has been criticized by various workers who contend that slight changes occur in the position of the retinal elements after the head is immersed in the killing fluid. In a series of controlled experiments, however, I could detect no post-mortem disturbances when the head was both bisected and cut from the trunk. 126 LESLIE B. AREY greatest care was not exercised, the retina proper easily separated from the pigmented epithelium. On the whole, the first method was preferred to the second because of the wrinkling of the retina that usually accompanied the use of the latter. Sections were cut 7 p to 10 p thick, and except in a few special cases only those passing through the region of the optic nerve were retained for examination. Preparations were stained with Ehrlich-Biondi’s triple stain or were double stained in Heiden- hain’s iron hematoxylin and a plasma counterstain. Ehrlich- Biondi in some instances gave excellent differentiation of all elements, while at other times it would show the capriciousness in producing satisfactory results for which it is notorious; iron hematoxylin gave uniformly good preparations. It often became necessary to bleach the pigment in order to study the visual cells, which would otherwise be masked by the partially or completely extended processes. The method em- ployed was essentially that of Mayer (W), in which nascent oxygen3 is the effective agent. The aim of the present investigation has been to determine the influence of light, temperature, anaesthetics and oxygen on the movements of the rods, cones, and retinal pigment in the normal and excised eyes of fishes and of amphibians. To Prof. G. H. Parker, under whose direction this research has been conducted, I wish to acknowledge my indebtedness for much inspiration and valuable suggestion. Advantage is taken of this opportunity to express appreciation for the facilities of the Zoological Laboratory placed at my disposal by the Director, Prof. E. L. Mark, and for many courtesies extended by him during my residence at Cambridge. 3. EXPERIMENTAL PART A. DETERMINATION OF ADAPTION TIMES Before extensive experimentation can be undertaken on the retinal elements, it is necessary to determine the various lengths 3 When potassium chlorate and hydrochloric acid interact, it is commonly said that nascent chlorine is the agent causing bleaching. As a matter of fact the reaction liberates free oxygen. MOVEMENTS IN THE VISUAL CELLS 127 of the time which they require in assuming the positions charac- teristic of light and of darkness. In most mses it is not easy to state definitely when adaption is completed, for the response becomes less vigorous as it nears the end and consequently the factor of personal equation is unavoidable. Light intensity and temperature (Dittler, ’07) represent variables which undoubtedly play a part in the determination of adaption time. No attempt was made to discover the exact r61e of either of these factors, although the general experimental conditions were kept approximately uniform during successive trials. The effect upon adaption time of a long or short preliminary subjection to light or darkness, has never been taken into ac- count, although such influences were asserted by Gaglio (’94). a. Retinal pigment Pergens (’96) found that after 2 minutes’ illumination the retinal pigment of Leuciscus began to expand. After 1 minute of darkness a noticeable contraction occurred, which was com- 20 minutes. Chiarini (’Mb), working on the same pleted in fish, came to somewhat different conclusions. He observed a sensible pigment expansion after direct exposure to sunlight for 1 minute, although complete light adaption necessitated a period of 1 hour. The reverse process of dark adaption was not initiated until the animal had been subjected to darkness for from 4 to 5 minutes, and a minimum of 1 hour was required to complete the contraction. When the retinal pigment of fishes has undergone a maximum expansion, it accumulates distally4 near the external limiting membrane, whereas the proximal portions of the cells are to a greater or less extent devoid of pigment (figs. 9, 10). Such a 4 The term ‘proximal’ as used in this paper will refer to movements toward the nuclei of a given cell, either pigment or visual. In like manner ‘distal’ will have reference to movements away from the nucleus. Since the distal movement in the pigment cells is the reverse of that in the rods and cones, an arbitrary nomenclature with reference to the eye-ball becomes confusing, while the ter- minology here suggested lends itself readily to descriptions of the moving parts. 128 LESLIE B. AREY condition of distal accumulation requires additional time after the ‘front rank’ of pigment granules has arrived at the position of maximal extension. In the determinations made by me, light adaption was not considered to be completed until the pigment was thus maximally expanded. The results obtained on the retinal pigment of fishes were as follows: Ameiurus Diffuse daylight 30 minutes, pigment incompletely extended 40 minutes, pigment fully extended, but homogeneously distributed 1 hour, maximal expansion Darkness 45 minutes, pigment about half contracted 1 hour, maximal contraction The movements of Abramis were more rapid, notwithstand- ing an apparently heavier pigmentation of the retina. Abramis Diffuse daylight 15 minutes, lightly pigmented processes three quarters extended. Most of the pigment at the bases of the cells Distal pigment accumulation 30 minutes, processes fully extended. begun 45 minutes, maximal expansion Darkness 20 minutes, pigment about half contracted 30 minutes, maximal pigment contraction The results of the dark adaption on Fundulus were less satis- factory, since the delimitation of the pigment in darkness is poorly defined. Fundulus Diffuse daylight 45 minutes, pigment fully extended; but little tendency exhibited towards distal accumulation 1 hour, maximal expansion Darkness 45 minutes to 1 hour, maximal contraction As regards the adaption time in the frog, Stort (’87) believed that in 1 hour of bright, diffuse daylight or in 4 hours of dark- MOVEMENTS IN THE VISUAL CELLS ness, maximal light- and dark-adaption of the pigment respec- tively was produced. Kiihne ('79) stated that complete dark- adaption occurred in 1 to 2 hours, this period corresponding to the time necessary for the regeneration of the visual purple. Chiarini ('04b) maintained that following an exposure of half an hour to direct sunlight one and one-half hours were needed to complete the adaption in darkness. Although no direct experimentation was made on the frog, the general experience gained from working with this animal leads me to suspect that 1 hour for light-adaption and 1 to 2 hours for dark-adaption are approximately the proper amounts. b. Visual cells Relative to the adaption times of the cone cells, Pergens ('96) stated that in Leuciscus the first visible shortening occurred after an exposure to light of 1 minute. According to his illus- trations, the cones were very much shortened after 2 minutes while those that had been subjected to light for 5 minutes were practically in the position characteristic for light. When light- adapted animals were introduced into the dark, a lengthening of the cones was evident after 1 minute and in 5 minutes the elongation was nearly complete, although it did not become maximal for 20 minutes. Pergens' method of allowing decapi- tated heads to remain in the light during fixation has been criti- cized by Garten ('07), and the later work of Pergens ('99) has likewise been questioned by Herzog ('05). These workers assert that it is entirely possible that the changes initiated by the action of light continue until the actual penetration of the fluidinto the eye fixes the retina. Certain experiments of Weiss, insti- gated by Garten, indicate that the action of light can influence the position of cones and retinal pigment after decapitated heads have been introduced into the fixitive. These results are in opposition to the statement of Chiarini ('04b) that light can- not be effective during fixation. In an attempt to avoid this source of error, at the completion of an experiment the light-adapted eyes were fixed in exceedingly 130 LESLIE B. AREY dim light. It is probable that this precaution was sufficient, for, as will be shown later, the movements of the cones of Abramis, the only fish worked upon, are not rapid and moreover no changes occur even when the excised eyes, immersed in water, are sub- jected to light or to darkness. Temperature is an important factor that must be considered in the adaption of cones. In anticipation of certain results that will be found in another part of this paper, it may be said that the cones of fishes are maximally extended at about 25°C. in the dark (fig. 27), and in the case of Abramis at least, they are also maximally shortened at 5°C. in the dark (fig. 25). Moreover, as temperature does not affect the length of the cones when they are under the influence of light, the animals may be kept at a temperature of 25°C. during an entire experiment and the re- sulting movement of the cones will then be solely traceable to conditions of light or darkness. The results on the cones of Abramis may be summarized as follows: Diffuse daylight 15 minutes, cones much shortened-perhaps two thirds 23 minutes, approximately the same condition as at 15 minutes 30 minutes, shortening not quite complete in most animals 45 minutes, maximal light adaption Darkness 13 minutes, cones somewhat extended-one third (?) 20 minutes, extension practically complete 30 minutes, maximal dark adaption The adaption times of the cones of Abramis are longer than those given by Stort for Leuciscus. This, in part, may be due to the wider range between the positions of maximal light- and dark-adaption which was produced by the aid of elevated temperature. Englemann ('85), working on the frog, was the first to discover that the movements of the cones were not accomplished in- stantaneously, but required definite periods of time. He also observed that elongation in the dark was a longer process than shortening in the light. My results on Abramis do not entirely support his latter view. At first the cones of this animal do MOVEMENTS IN THE VISUAL CELLS 131 respond more actively when stimulated by light, but a longer time is required to complete the process of light-adaption than the reverse changes in the dark. No experimentation seems to have been performed upon any animal to determine the adaption time of the rod cells. Ameiurus was selected for these tests, for the rods differ greatly from those which are characteristic of fishes in general. Instead of the slender elements 1.5 p to 2.0 p in diameter, which, for example, are found in Abramis (fig. 25), the rods in Ameiurus (fig. 31) are robust and resemble more closely those of the frog (fig. 35). The barrel-shaped ellipsoid measures about 4 p in either dimen- sion, and the width of the outer membrane is the same. With this can be compared the width of the outer member of the rods in the frog, which in my preparations of R. pipiens measured, for the most part, 5 p, although Howard ('08) states the width as 6 p, and H. Miiller ('56) as from 6 p to 7 p.L The species was not mentioned by either of these writers. Unlike the cones, the rods of Ameiurus in the dark form a more or less even row close to the external limiting membrane (fig. 31), while in the light the myoid elongates carrying the ellipsoid and outer member far up into the pigment layer (fig. 30). The extent of these positional changes may be judged from measurements of rods in darkness and in light which give ex- treme values of 70 p and 7 p respectively for the length of the myoid. I know of no fish in which rods of this size have been described, although Garten ('07) makes particular mention of the pike as possessing 'grosse Stabchen.' It is evident that the large size and the extensive positional changes which the rods of Ameiurus undergo make them especially favorable for physiological experimentation. The effect of temperature upon the length of the rods is com- paratively slight, hence the following determinations on the rods of Ameiurus were conducted at room temperature. 6 Perhaps the fact that my measurements were made on dark-adapted rods accounts for this discrepancy, for in rod cells the outer member is said to become longer and slenderer in darkness than in light. 132 LESLIE B. AREY Diffused daylight 30 minutes, rods two-thirds extended 45 minutes, maximal light adaption. (Cones also light-adapted) Darkness 15 to 20 minutes, rods, in most cases, almost completely shortened 30 minutes, maximal dark adaption. (Cones still in position of light adaption) The quicker response of the rods in darkness than in light is noteworthy. The rod shortens in the dark, the cone in the light; since in both cases the process of shortening is more vigorous than the lengthening, it would appear that the contractility of either type of cell is the responsible factor and the relative effici- ency of light and darkness is not primarily involved. In the last analysis, however, the situation may not be reducible to such simple terms. If these responses are merely the expression of the action of light and darkness on the protoplasmic myoids, why should the direction of movement of the two elements be opposed? A discussion of this phase of the problem will be found in another place. I attempted no experimentation upon the cones of the frog. Angelucci ('90) stated that after an exposure to candle-light for 5 minutes, the cones were strongly retracted, although other experiments of his do not seem to support this conclusion. Her- zog ('05) found that at medium light intensity complete light- adaption occurred in 24 minutes. The most surprising discovery in this series of determina- tions, taken as a whole, was the length of time required to com- plete the adaption of the rod and cone cells in comparison with the retinal pigment. From the results of earlier workers, 1 had expected the positional changes of these cells to be com- pleted in a few minutes, hence the actual values obtained were wholly unlooked for, and were only accepted after many repe- titions of individual experiments, MOVEMENTS IN THE VISUAL CELLS 133 B. EFFECT OF TEMPERATURE (NORMAL ANIMALS) a. Retinal pigment No investigations have hitherto been made to determine the effect of temperature on the retinal pigment of fishes, whereas several workers have used the frog for experimentation of this kind. The problem considered here was to determine the re- sponse of the retinal pigment of normal fishes and amphibians to various temperatures, both in light and in darkness. 1. Fishes. Experiments in the light were performed in the following way. Light-adapted6 fish were placed in large battery jars close to north windows where they received strong diffuse daylight; sheets of white paper were always placed under the jars to aid reflection.' The highest temperature to which it is safe to subject fishes is about 28"C., although by gradual elevation a somewhat higher temperature can be withstood (Loeb and Wasteneys, '12). A low temperature that did not vary beyond the limits of 3" and 5°C. was produced by intro- ducing small pieces of ice directly into jars with the fish. At this temperature fish are for the most part inactive, the respiration rate decreases and they remain quietly at the bottom of the jars. At the end of an experiment, which was never less than three hours long, the eyes were excised and immersed in fixing fluid in the light.8 During the earliest trials retinas from the same fish were compared, eyes being subjected to the extreme temperatures in 6 The terms 'light adaption' and 'dark adaption' as used throughout this paper imply that the animals had been previously subjected for a minimum of 4 hours either to bright diffuse daylight or to total darkness. The possibility of a dark background influencing the distribution of retinal pigment was considered. Such a visual control, if present, would correspond to the known r81e of the eye, as determined by Pouchet ('76) and others, in mi- mals which adapt their body color to the immediate environment. A series of careful comparisons, however, failed to show any recognizable differences be- tween the pigment distribution in the retinas of fishes that had been kept over dark or light backgrounds. In all work which involved the use of temperature, the fixing fluid was kept at the same temperature as that at which the experiment had been performed. Such treatment eliminated a possible source of slight error. 134 LESLIE B. AREY successive experiments. At first this seemed to be the correct procedure, but rigorous controls showed that such precautions were unnecessary. When interpreting the results of experiments conducted in the light it is not the absolute amount, but rather the relative distribution of pigment that serves as a basis for decisions. In experimentation in the dark, absolute differences in the degrees of pigmentation could give rise to errors in judg- ment, for the pigment, gathered into compact masses in the cell ‘cups’ might mask or apparently reverse the effect of temperature. Fortunately, however, the retinas, as judged from the expanded light condition seem, on the whole, to be very equally pigmented and the width of the contracted pigment zone, therefore, gives a fair index of the effect of temperature in the dark. Wh%n work- ing in the light especially, it.was found to be very desirable to have the experiments at contrasted temperatures conducted simultaneously in order that advantage might be taken of iden- tical light conditions, for as will be shown, light intensity is an important factor in obtaining the maximum expansion of pigment. as follows. Experimentation in the dark was conducted A fireless cooker, lined with black paper, was used as a dark cham- ber, on account of the minimal loss of heat incurred by it during the course of an experiment. If such an apparatus be previously brought to the temperature of the introduced jar of water, an experiment can be continued for several hours without further attention. In all determinations mentioned in this paper which were conducted in the dark, precautions were taken against the possible influence of light during the few seconds necessary for excision and transference of eyes to the fixative. Along series of careful comparisons showed identical results whether the operation was carried out in total darkness or in light of just sufficient strength to permit the operator to see the animal and his instruments. Indeed, the results obtained by operating in an ordinarily lighted room showed no recognizable differ- ences in either pigment, rods, or cones from those secured by working in darkness. A detailed description follows of the conditions found in each of the four fishes: MOVEMENTS IN THE VISUAL CELLS 135 1. Ameiurus. At 25°C. in the light (fig. 2), the characteristic position of the expanded pigment is in a broad band about 95 p wide, which extends nearly to the external limiting membrane. The pigment granules are evenly distributed and show no ten- dency to aggregate distally. At 15°C. the condition is very similar to that just described. The pigment, on the whole, tends to be homogeneously dis- tributed, although in many retinas at this temperature there is a slight distal accumulation. The disposition of pigment at 5°C. is markedly different (fig. 1) for it migrates to an extreme d'stal situation and forms a dense zone, approximately 30 p wide, close to the external limiting membrane, although the pigment of fishes and amphib- ians, under normal conditions, never actually touches this membrane. Between this heavy pigment-mass and the bases of the cells lie scattered granules, but the intervening space, nevertheless, appears relatively devoid of pigment. This ex- treme condition is best produced on the brightest days, and it is impossible to obtain as complete a migration on cloudy days, regardless of the temperature. On the other hand, the uniform distribution characteristic of incomplete expansion at 25"C., is independent of the intensity of diffuse daylight. This would suggest that a high temperature is more efficient than light in the regulation of pigment distribution, and that cold, that is, the absence of heat, merely allowed light to act unrestrained. Light and high temperature, then, are antagonistic in their effects. The results at 5", 15", and 25°C. are, in a way, what might have been expected. A temperature of 15" to 25°C. probably represents the greatest average warmth to which the animal is subjected in nature; this range from 15" to 25"C., then, repre- sents the limits of what may be called a warm environment for the animal. In the same way from 0" to 10°C. may be called a cold environment, and from 10" to 15°C. a neutral environment, neither particularly warm nor cold. Hence it is not surprising that the results at 15°C. are more similar to those at 25°C. than at 5°C. At 10°C. the distribution of pigment approximates rather more closely that at 5°C. A curve, therefore, obtained 136 LESLIE B. AREY by plotting temperatures as abscissas and the quantitative amount of distal migration as ordinates would show a gradual slope from 0" to lO"C., from 10" to 15°C. a rapid drop and from 15" to 25°C. a nearly horizontal but slightly sloping line. The results of experiments performed in the dark, where the is highly contracted, are usually not as clear cut as pigment reason for this is because it is im- those just described. The possible to see the qualitative distribution of the pigment granules and hence decisions regarding the effect of temperature must depend largely on measurements of the width of the narrow a criterion, as has previously been pigmented layer. Such pointed out, is open to the criticism that individual eyes may vary enough in the absolute amount of contained pigment to disconcert judgments concerning the effect of temperature. After having studied a great number of preparations, I do not believe that such an unequal pigmentation is in truth a factor that warrants serious consideration. However this may be, an obvious precaution consists in the prolonged repetition of each type of experiment. It may be said that in the course of my experimentation on the effect of temperature on normal fishes alone, over 200 retinas have been examined. The evidence obtained from Ameiurus, was more conclusive than that from any of the other fishes, with the possible excep- 4) the pigment forms a densely tion of Carassius. At 25°C. (fig. contracted layer, the mean width of which is about 25 p. In contrast with this is the condition at 5°C. (fig. 3), where the cells have short pigmented processes, the total extent of which is approximately 38 p. The differences apparent at these two extremes of temperature were so slight in comparison with the much greater variation in the light, that thorough experimenta- at the intermediate temperature of 15°C. was not attempted, tion it was sufficiently demonstrated that the results at although this temperature do not vary to any great extent from those at the two extremes, and probably more closely approximate the highly contracted condition at 25°C. The results of some of the temperature determinations were not conclusive. Reference has already been made to the fact that MOVEMENTS IN THE VISUAL CELLS on dull days maximal expansion in the light was hard to obtain. Among fishes in general, more doubtful cases occurred in ex- periments conducted in the dark than in the light, yet in all such cases the uncertainty merely lay in deciding between two nearly equal conditions, while in practically no instance was there evidence of a definite reversal whereby a greater distal migra- tion occurred at 25°C. than at 5°C. 2. Fundulus. The conclusions reached from the study of Fundulus, as well as from the other fishes, are similar to those given for Ameiurus, but each fish shows individual peculiarities in the disposition of the pigment and these will be briefly described. In the light the pigment of Fundulus tends to migrate to a great extent forming a broad zone at the distal ends of the cells, much denser and more sharply defined than in Ameiurus. Be- tween this zone, which has a width of 30 p, and the bases of of pigment at the cells there is a clearer area, almost devoid 5°C. (fig. 5), while at 25°C. (fig. 6) this region contains a con- siderable amount of evenly distributed pigment granules. In the latter case, however, the pigment still forms a closely aggre- gated zone distally, although it is reduced to a width of 17 p. As in Ameiurus the condition at 15°C. more closely resembles that at 25°C. than at 5°C. A peculiarity in preparations of the light-adapted retina of Fundulus (at least with Perenyi’s fixation) is that at the higher temperatures the pigment extends in columns from base to periphery of individual cells (fig. 6), while between such columns of adjacent cells are elongated areas free of pigment but taking the plasma stain. Examination under high magnification does a casual not show the presence of excessive shrinkage,. although observation might suggest that this had occurred. Where the pigment is aggregated at the base and periphery of the cells, cell boundaries are not distinguishable and the pigment appears as homogeneous masses. The separate columns connecting these two continuous zones give the whole an appearance not unlike a ladder with rungs set very close together. THE JOURNAL OF COMPARATIYE NEUROLOGY, YOL. 26. NO. 2 138 LESLIE B. AREY In the dark the contraction is very incomplete and some- times tends to give rise to a distal accumulation of pigment which faintly resembles the distribution in the light, although such a condition is not constantly present as a characteristic of dark adaption. It is impossible to state the cause of this pigment massing. It may be due to a greater activity at the distal ends of the cells in producing a contraction, or what would bring about a similar end result, a contraction of pigment en masse. Measurements were taken of preparations at the extreme temperatures in the dark, the degree of variation being greater than in any of the other fishes studied. Thirteen retinas at 5°C. (fig. 7) showed the pigment to be extended a mean dis- tance of 20.4 divisions of the ocular micrometer, whereas fif- teen retinas at 25°C. (fig. 8) had a corresponding value of 14.4. 3. Abramis. In the light the condition in the eye of this fish is somewhat similar to that in Fundulus. The pigmenta- tion is very heavy, forming a broad zone near the external limit- ing membrane; between this distal zone and the base of the pigment cells pigment granules are also present, the density of pigmentation depending on the temperature. At 5°C. (fig. 9) the distal zone is wider than at 25°C. (fig. 10) in the ratio of 50 p to 38 p, while the proximal area is much less heavily pigmented than at 25°C. Although each area is sharply defined, the one appears to grow at the expense of the other. It was not possible to get so complete a distal accumulation of the pigment as in Fundulus and, as will be presently shown, the extent of migration in Fundulus is still less complete than that in Carassius. The dark phase is one of great contraction, and although some retinas show evident differences at the extreme tempera- tures, yet throughout the whole set judgment of the eye has to be supplemented by actual measurements. Such measure- ments show that the mean expansion at 5°C. (fig. 11) exceeds that at 25°C. (fig. 12), the values being 30 p and 20 p respectively. 4. Carassius. The eye of the goldfish is more heavily pig- mented than those of the three other fishes. -4t 5°C. in the MOVEMENTS IN THE VISUAL CELLS 139 light (fig. 13) all the pigment is located distally forming a broad, dense zone. Between this band and the base of the cells is a narrow clear area in which scattered pigment granules are visible only with the aid of high magnifications-for practical purposes it may be said to be free of pigment. The situation at 25°C. in the light (fig. 14) is variable. In some cases it closely simulates tfiat at 5"C., although the clearer space is always relatively more heavily pigmented ; in other instances, the pigment is uniformly distributed from the proxi- mal to the distal extent of the cells. The contraction that usually occurs in the dark was less pro- nounced in Carassius than in any fish heretofore described; in fact in some eyes that had been subjected to a low temperature, the actual breadth of the pigment layer nearly equalled that of a light-adapted eye. The relative expansion at the extreme temperatures in the dark, however, leaves no doubt that at 5°C. (fig. 15) the contraction is less than at 25°C. (fig. 16). Measurements (in terms of the divisions of an ocular micrometer) taken from eight eyes at 25°C. and ten eyes at 5°C. gave re- spective mean values of 8.0 and 12.1. From observations on these four genera certain generaliza- tions are suggested. The degree of pigmentation in the eye of Ameiurus (figs. 1, 2) is much less extensive than in the other fishes, as a comparison with Carassius (figs. 13, 14) shows in a striking manner. The three other fishes, however, offer better opportunities for comparison since their pigmentation is more nearly equal. At 5°C. in the light the pigment of both Carassius and Fundulus (figs. 13, 5) migrates distally to such an extent that a proximal zone, devoid of pigment, is created. Moreover, the dark phase of the pigment, in both these animals (figs. 16, S), is one by no means extreme when compared with the highly compact layer in Abramis and Ameiurus (figs. 12, 4). In the last named fishes, on the contrary, it is impossible under the most favorable conditions of light and temperature to obtain the proximal clearer area entirely free from pigment; correlated with this absence of complete expansion is the high degree of contraction which is evoked in the dark. 140 LESLIE B. AREY It does not seem probable that quantitative differences in the degree of pigmentation can be the cause of such relations, for if the relative amounts and the distribution of pigment in Fundulus and Abramis be compared at 5°C. in the light (figs. 5, 1) and at either temperature in the dark (figs. 7, 3) it must be admitted that quantitative differences do not adequately explain the conditions that exist. A clearer insight is gained if these responses are viewed from the standpoint of cell organization. We can think of two gen- eral types of pigment cells in which the pigment distribution is correlated with the behavior outlined above. Thus, in one type, at the distal end; such a the pigment would tend to remain cell would show maximal expansion but relatively incomplete contraction. In a second type of cell, in which the pigment aggregates more proximally, maximal contraction but incom- plete expansion would be accomplished. Although other species of fishes should be studied before a final conclusion is reached, this set of relations may be general. The correlation can be stated, at least tentatively, as follows-the highest degrees of expansion and contraction in the retinal pigment of fishes are mutually exclusive in the same retina. 22. Frog (aduZt and larva). As in many other lines of physiologi- cal work, the frog has been used to a large extent by investigators of retinal physiology. Ewald und Kuhne ('78) performed the first experiment in which the position of the retinal pigment of the frog was shown to be dependent upon temperature. According to their account, after 2 hours' immersion in ice water in the dark, the pigment showed a distribution similar to that obtained at 17"C., which may be called a state of contraction.Y When, however, frogs were subjected to 30°C. for 2 hours, an expansion occurred in which lightly pigmented processes were said to extend even to This, however, does not coincide with an earlier statement ('77, p. 250) of the same authors, "Vor allem ist die Temperatur von ausserordenlichem Ein- flusse . . . . Frosche, welche 1-2 Stunden in Eiswasser gehalten wurden, liefern schwarze Netzhaute, indem das ganze Epithel mit ausschldpft, und nicht vie1 besser verhalten sich die Priiparate von solchen, die hei 5"-10"C. im Dunkeln verweilten." MOVEMENTS IN THE VISUAL CELLS 141 the external limiting membrane. These workers were primarily interested in discovering under what conditions of temperature an accumulation of pigment in the rod area could be avoided, for their investigation dealt with the visual purple. Since the frogs used for these determinations were treated with curare in order to produce pigment relaxation, and, moreover, since these animals were in an oedematous condition as a result of this poisoning, it is evident, as Herzog ('05) pointed out, that much weight can not be given to their conclusions alone. To Gradenigro ('85) belongs the credit of having performed the first temperature experiment upon the retinal pigment of normal animals. He introduced a dark-adapted frog into a dry, darkened chamber and removed the whole to a dark room. A temperature of 30°C. was maintained until heat rigor set in, when, on examination, the pigment was found to be in a condi- tion of maximal light expansion (fig. 19). Gradenigro's results were confirmed by Angelucci ('90) and by Fujita ('11). Herzog ('05), without knowledge of Gradenigro's contribution, undertook a detailed study of the relation between temperature and pigment distribution. His results not only corroborated those of Gradenigro, but also established the additional facts that at low temperatures (0"-14°C.) in the dark the distribu- tion of pigment is identical with that at high temperatures (fig. 17), while only between 14" and 18°C. in the dark (fig. 18) is maximal pigment contraction obtained. His experiments were performed in the following manner. Dark-adapted frogs (R. temporaria and R. esculenta?) were placed in a heating chamber at an initial temperature of 20"C., the introduction of the animals cooling the apparatus to 17"-18"C. Progressive heating raised the temperature in 15 minutes to 24"C., in 30 minutes to 32"C.., in 45 minutes to 37"C., and in one hour to 39-40°C. At each fifteen minute interval frogs were removed and their eyes prepared for microscopical examination. At 24°C. the position of the pigment was not essentially dif- ferent from the normal state of maximal contraction, although delicate fringed processes did extend towards the external lim- iting membrane. It is probable that such an experiment did 142 LESLIE B. AREY not fairly test the effect of this temperature on pigment migration. One could hardly expect the body temperature of the animals to become adjusted to that of the apparatus in such a short time, especially since the heating was progressive and the final tem- perature was not realized until the end of the experiment. At 32°C. the pigment had extended to the maximal distance but the distribution was nearly homogeneous. A condition of extreme expansion occurred at 37°C. A dense massing of pigment near the external limiting membrane masked the rod ellipsoids completely, while the outer members of the rods were nearly free from pigment. When the temperature was raised to 39” of 40°C. clonic spasms occurred which ended in death; the pigment, nevertheless, retained the same position as at 37°C. A second series of experiments, in which frogs were cooled in a refrigerating apparatus, showed that a subjection to 0°C. in the dark for 2 hours produced incomplete expansion, while after 3 hours the pigment was distributed in a zone of maximum breadth, but with only a slight tendency toward distal massing. This discovery, which was Herzog’s most interesting contribu- tion, is not only in disagreement with the commonly quoted result of Ewald und Kiihne’s earlier work, but also has not been substantiated by the recent investigation of Fujita (’ll), who. however, states that high temperature does induce pigment expansion, as the other investigators have all maintained. Fujita tried the effect of ow temperature on only four animals, the duration of his experiments ranging from 30 minutes to 6 hours, yet he drew the following positive conclusion (p. 170): “Das Resultat war in allen Fallen das gleiche: ich konnte keine Hell- stellung konstatieren. Die Zapfen waren nicht kontrahiert , das Pigment nicht vorgewandert .” Since all these results on the frog’s retinal pigment are not only fundamentally different from those found by me in fishes, but also have no parallel in the movements of vertebrate and invertebrate melanophores, and since, moreover, there is no general agreement concerning the effect of low temperature, a thorough reinvestigation of the problem seemed to be needed. MOVEMENTS IN THE VISUAL CELLS 143 My first effort, therefore, was to repeat Herzog's work using apparatus and methods that essentially agreed with his, in order to ascertain whether identical results would be realized. If the movements of the frog's retinal pigment are really ex- ceptional among other lower vertebrates, such responses have considerable theoretical as well as incidental interest. X cubical cage, made The following apparatus was devised. of fine wire netting with a cover of the same material, was sup- ported by uprights inside a large battery jar which was fitted with a glass cover perforated by a small hole to allow slow dif- fusion of air. The wire cage, made to receive the frog, did not come in contact with any part of the surrounding glass receiver which was to serve as a constant temperature chamber. The glass receiver sat upon a platform in a large cylindrical metal tank. A thermometer passed through the cover of the tank and also through the cover of the temperature chamber into that chamber itself. A funnel connected with a rubber tube to exclude light was fitted into the cover of the tank and served for introducing water into the tank. A dark-adapted Tests were conducted in the following way. frog was wiped dry and placed within the wire cage10 inside the constant temperature chamber. Previously the chamber had been brought to the desired temperature by one of two simple methods. If the effect of heating was to be studied, the metal tank was partially filled with water at the appropriate temperature and the whole system allowed to adjust itself until the ther- mometer inside the constant temperature chamber registered the required degree of warmth. After the frog had been in- troduced, the temperature could be regulated without opening the apparatus by admitting warmer water through the funnel and drawing off an equivalent amount from the bottom of the tank. If, on the other hand, a temperature near the freezing lo Hersog lays much stress on the facts that the animal's body was never in contact with solids other than the wires of the cage, and that the body was always wiped free from secretions or excretions at the commencement of an experiment. He seems to fear lest there should be a chemical stimulation due to heated secre- tions or excretions that would affect the results in case these precautions were not followed. 144 LESLIE B. AREY point was desired, the outer chamber of the tank was filled with a mixture of ice and water; if a sufficient amount of ice was provided a temperature of from 3" to 5OC. could be maintained, without further attention, throughout the whole experiment. At the expiration of the time allowed for temperature adaption (usually 3 hours), the apparatus was placed in the dark or in weak red light and the frog's eyes were removed and fixed in darkness at the same temperature as that at which the experiment had been conducted. In the course of these tests nearly 100 eyes were examined, yet the general results obtained were identical with those de- scribed by Herzog. At 3°C. and 33°C. (figs. 17, 19) the pig- ment was expanded approximating the condition characteristic of light; between the temperatures of 14°C. and 19°C. (fig. 18), however, the pigment was contracted to a narrow compact layer. It should be noted that 14°C. and 19°C. may not represent the limiting temperature at which pigment contraction occurs, although Herzog states this to be the case; no attempt was made by me to determine these intermediate temperature-limits. ,4t low temperatures the expansion of pigment was generally not as complete as at a high temperature or in the light, and there was often considerable variation in different parts of the same retina, yet the general result was one of unmistakable expansion. In some cases, however, there was little or no evidence of a pigment expansion at the lower temperature, yet such instances were comparatively rare. Although the cause of discrepant results of this kind is not evident, they perhaps furnish additional proof for the nervous control of the frog's retinal pigment, as many workers maintain (vide infra). It seems probable that Fujita was unfortunate enough, in the few experiments which he performed at a low temperature, to obtain nothing but this lack of typical results, although in my own work the occurrence of such anomalous cases was always sporadic. A careful comparison was made of the results obtained at 3°C. and 33°C. No constant difference in the amount of migration could be detected, although Herzog states that at 37°C. the distal migration is greater than that obtained at low temperature. MOVEMENTS IN THE VISUAL CELLS 145 Hence we may conclude that the condition found in the frog is unlike that found in the fishes. It should be noted, however, that between certain limits the two animals show similar ten- dencies in their pigment responses. These limits are approxi- mately 0" to 19°C. in the dark for the frog, and 0" to 28°C. in either darkness or light, for the fishes. Since the tendency of pigment migration under the influence of temperature agrees between the limits of 0" to 19°C. for the dark adapted frog and 0" to 28°C. for the fishes either in light or in darkness, the query may be raised-is it not possible that if the fishes were subjected to higher temperatures a reversal of the temperature effect would occur in which an expansionof the pigment would again be found as in the frog? The fact that Herzog found but slight differences at 18°C. and 24°C. increases this suspicion. I am convinced, however, that a tendency to- ward such a response does not exist in the retinal pigment of fishes even to the slightest extent. In the first place, as stated before, prolonged heating of the frog's retina at 24°C. would be likely to produce more striking changes than Herzog obtained. Furthermore, a few experiments in which the temperature of fishes was raised to 30°C. and over failed to show, in either light or darkness, anything beyond the. characteristic response of less complete expansion than at the lower temperature. The extent to which the retinal pigment of the frog and of fishes moves under the influence of temperature differs in a high degree. Among the fishes the differences are small and amount to little more than a redistribution of pigment in the dark and light phases respectively, whereas in the dark-adapted frog varying temperature induces the whole range of pigment re- sponse usually occasioned by light or darkness. This further indicates that the nature of the response in the two kinds of pig- ment cells differs fundamentally. In the fishes probably the response is through the direct action of temperature on the cell protoplasm, while in the frog the pigment migration may be produced indirectly through the intervention of the nervous system. 146 LESLIE B. AREY All the experiments of previous workers on the effect of tem- perature have been performed in the dark and I, therefore, set about to discover what results would be obtained in the light. The only change in the apparatus from that previously described was that a 20 litre jar replaced the metal tank; this, when filled with water at the desired temperature, performed the necessary heating function while its transparency did not interfere with the entrance of light into the inner chamber. Experiments were made at the same temperatures as in the dark-3" to 5"C'., 16°C. and 33"C., but the results were, for the most part, condi- tions of uniform expansion independent of temperature. It was observed that when frogs were subjected to a low temperature they became quiescent and tended to keep their eyelids closed. Although the lower lid (the only one which is movable to any extent) is more or less transparent, the possi- bility of its influencing the results led to its removal in a number of instances; no difference, however, was obtained by the ob- servance of this precaution. From these results, therefore, we conclude that in darkness, temperature is the controlling factor, while in the light temperature is subordinate to the stronger stimulus, light. At this juncture a doubt arose as to the exact temperature the frog's retina was experiencing while in the apparatus. It is well known that at the surface of the frog's body rapid evapora- tion can take place; hence it is perfectly conceivable that the rate of evaporation in the temperature chamber might be such as to keep the body temperature for some time considerably below that of the surrounding air. This possibility was first checked by taking the oesophageal and rectal temperatures of animals that had been subjected to various temperatures in the apparatus for several hours. The recorded temperature, how- ever, was nevei found to vary more than a fraction of a degree from that of the air in the containing chamber. To be absolutely certain on this point, a prolonged set of ex- periments was made in which the frogs were immersed in water media of appropriate temperatures. It is certain that after a short time the animal, as a whole, must assume the temperature MOVEMENTS IN THE VISUAL CELLS 147 of the medium irrespective of activities at the surface of the body. The apparatus for this verification consisted merely of a large battery jar, a sheet of coarse wire gauze and suitable weights. The jars were filled with water to within a quarter of a centimeter of the top, and the gauze, held in place by weights, served as a cover. This device worked in the following manner. Animals coming to the surface to breathe could only get their nostrils above water, the rest of the head and body remaining submerged, hence, in a short time the body temperature of the frog neces- sarily approximated that of the surrounding medium. At 3°C. the body temperature of the animal quickly fell, the respiration rate was reduced until it practically ceased and the bodily activities diminished until the frogs, for the most part, remained quietly at the bottom of the jar, although some animals would occasionally swim to the surface to breathe. At 33"C., on the contrary, the frogs were very active and had to return at short intervals to the surface, where they would sometimes remain for several minutes clinging to the netting. A series of experiments was performed both in darkness and in light. In the dark nothing new was learned beyond the con- ditions already described. In the light, the first experiment showed a state of extreme pigment expansion at 3"C., which of Ameiurus under similar conditions. was comparable to that at 16°C. and 33"C., on the contrary, showed the The other trials, a short pigment uniformly distributed. Another experiment, time after, gave the same result at 3°C. but not so decisively. The possibility of discovering a similarity in the pigment re- sponses of frogs and fishes in the light, led me to repeat these ex- periments many times without, however, again obtalning similar results. If extreme pigment expansion occurred at 3°C. it would be interesting from another standpoint since Herzog reported a similar condition, in darkness, at the highest temperatures which these animals can withstand. Occasionally during experiments both in light and in dark- ness, an anomalous condition arose whereby the distribution 148 LESLIE B. AREY of pigment in one part of the retina was markedly different from that in the remaining portions.11 Such conditions may have been due to a variety of disturbing factors, Angelucci (’90) has recorded noises, unilateral pressure on the eyeball and mechanical or electrical stimulation of the body as causing the migration of pigment in dark-adapted animals. Herzog (’05) likewise states that a frog tied up for 24 hours in the dark showed the pigment in the light position. A whole series of experiments and observations are on record to show motor control of some sort not well understood. It certainly is evident from a com- parative study of pigment in other forms, that is, in the retinas of fishes as well as in vertebrate and invertebrate melanophores, that the situation in the frog is entirely unlike that in any other animal concerning which we have data. Herzog explained the temperature responses of the frog’s pigment in the following way. If the effect of temperature is purely physical, its action presumably consists in accelerating or retarding chemical processes in the protoplasm of the pigment cell. Since, however, the movements of the dark-adapted pigment are not directly correlated with the temperature grad- ient, a physical action of temperature is probably not responsi- ble for the observed phenomena. If, on the other hand, it is assumed that the response of the pigment involves the principle of ‘specific energies,’ then any positive stimulus, acting through the nervous system, will cause a pigment expansion, and thus a satisfactory explanation for the known facts is furnished. In connection with the special case offered by the frog an in- teresting speculation arises as to the kiiid of pigment responses shown by the frog larva. The larva, in a general way, is com- parable to a fish; at least it may be said that the larval stage recapitulates certain conditions persistent in the adult fish. Is it not possible, therefore, that under the action of temperature the pigment of the larval frog will show a distribution similar to 11 A somewhat similar lack of consistency was also noted by Fick (’go), in his attempts to obtain maximal contraction in dark-adapted eyes. He concluded, however, that inequality in the pigment distribution was characteristic of dark- adapted retinas. MOVEMENTS IN THE VISUAL CELLS 149 that in fishes? The material, on which. an answer to this ques- tion was sought, was the larva of the bullfrog (R. catesbiana). Animals were obtained during the month of April, 1914; these, of course, represented larvae hibernated from the previous season, since two or even three years are required to complete the meta- morphosis of this species. A few experiments were also made on animals obtained in November, 1914. From the larvae procured, two size limits were selected for experimentation. The smallest larvae had a total body length of 4.5 cm., the hind legs of such animals not beingvisible; the 7.0 em., and the hind largest larvae had a body length of legs were developed as two small buds with the digits just differentiated. In neither the 4.5 cm., nor the 7.0 cm. animals were the eyes as deeply pigmented as in the adult of R. pipiens. Of the two sizes, the 7.0 cm. larvae had the pigment more highly developed, and consequently better differentiation was obtained at the various temperatures with these, than with the smaller animals. The 4.5 em. larvae at 3", 26", and 32°C. in the dark showed the pigment in an expanded state (cf. figs. 20, 22) but not in a firm zone of uniformly distributed granules. On the contrary, the cells displayed pigmented processes that seemed to be more or less independent of each other; the appearance of the zone being that of a more uniform base with a fringe of pigment extending distally from it. The degree of expansion at 26°C. was at either 3°C. or 32"C., although at neither distinctly less than of the extreme temperatures was the pigment as fully extended as in the light. At 16"C., however, a striking difference was found (cf. fig. 2l), for the pigment lay contracted in a narrow compact layer near the choroid. The 7.0 em. animals gave results (figs. 20, 21, 22) quite similar to those just described, although the contrasts at the various temperatures were considerably sharper due to the heavier pig- mentation of the eyes at this stage. In these larvae, therefore, the behavior of the retinal pigment to temperature is identical with that characteristic of adult frogs. Since these animals were always immersed in water, there is no 150 LESLIE B. AREY question concerning the correspondence of their body temperature and that of the surrounding medium. What would be discovered in a study of earlier stages I can I suspect great difficulty would be encountered in not say, but interpreting the results due to incomplete pigmentation, for the differences at various temperatures in the 4.5 em. tadpole, although fairly well marked, showed much less contrast than those exhibited by the 7.0 cm. animals. 3. Necturus. A comparison between the frog and some uro- dele suggested another interesting problem. Is the condition exhibited in the frog restricted to anurans or is it common to the whole group of amphibians? This query becomes all the more pertinent when it is recalled that urodeles are not in the direct line of ascent to the anurans; that is, they are not amphib- ians which have never gone beyond the water inhabiting stage, but are more probably a group that were once land animals and have again returned to the water as a secondary adaption. The common mud puppy, Necturus maculatus, was chosen because of the ease with which it is procured and kept in cap- tivity. These animals were treated according to the technique used for fishes, hence a temperature higher than 28°C. was n ot at tempted . The first experiments performed were to secure typical light- and dark-adapted retinas in order that some basis of compari- son might be had. The results were by no means as striking as one might wish. In both cases the pigment was extended, not in a band with even contours, but generally in large conical proc- esses from the individual cells, which, like other cells of Necturus, appear to be very large; these conical processes surround the distal ends of the outer segments of the rods. In the light, the pigment was usually somewhat more ex- tended than in darkness. The processes were not of uniform length but mean measurements may be expressed by the values of about 38 p in the light and 30 p in the dark,-a condition of slight contrast when compared with fishes or the frog. The presence of a certain amount of migration in Necturus has been previously noted by Howard ('08), who took advantage of MOVEMENTS IN THE VISUAL CELLS 151 the contraction in the dark in his study of visual cells. This situation is comparable to t8hat found in Triton where movenients of the pigment through the influence of light were discovered by Angelucci ('78), Stort ('87) and Garten ('07). The extent of pigment migration in this animal is described as being very limited by Garten (p. 70) who says: ". . . . dieselbe ist aber hier unvergleichlich vie1 schwacher als bei vielen anderen niederen Wirbelthieren. " In an examination of the effects of various temperatures on dark-adapted eyes, however, no constant differences were noted. In some instances the processes of the cells seemed to be less heavily pigmented and the bases more heavily pigmented at 15°C. than at the two extreme temperatures, but this was by no means constant. At least, it can be said that there is no marked contraction of the pigment in the dark at any temperature. The evidence from Necturus and the limited pigment migration in Triton conclusively prove that the pigment responses typical of the frog are not common to all amphibians. It is probable, therefore, that such peculiarities as were described for the frog have been developed solely within the anuran group. b. Visual cells Although the myoids of both the rod and cone cells of fishes are capable of a high degree of contractility (a 90 per cent re- traction occurs in some instances), the effect of various tempera- tures on these cells remains untried up to the present time; in fact, the frog is the only animal upon which such work has been attempted. Gradenigro ('85), Angelucci ('84b), Herzog ('05), and Fujita ('11) found that warming produces the same effect on the cones of the frog as does light. Herzog also stated that cooling to 0°C. likewise caused the cones to shorten, although Fujita denies that this occurs. There is no record of any attempt to determine the effect of temperature upon the rods of vertebrates beyond the statement of Gradenigro ('85) that at 30°C. the rod of the frog shortens as in light. 152 LESLIE B. AREY The object of the work upon visual cells to be described in this section parallels that stated for pigment, that is, to deter- mine the effects of various temperatures on the myoid of rod cells and of cone cells in normal fishes and amphibians. The apparatus and technique employed were similar to those which were used in the experimentation upon pigment. Par- ticular care was exercised at the termination of experiments conducted in the dark to guard against the action of light on the highly sensitive visual cells. In the fishes, measurements of the cone myoid were made from the external limiting membrane to the proximal edge of the ellipsoid, and in the frog, from the external limiting membrane to the proximal side of the oil drop which is situated at the distal end of the ellipsoid. The lengths of the rods were measured from the junction of the inner and outer members to the external limiting membrane. Each value given in the tables represents the mean measurement, in niicra, of many (12 to 24) individual elements. 1. Fishes. (1) Ameiurus. Of the four fishes studied, the cones of Ameiurus, in many ways, gave the least satisfactory results. These elements are not located at uniform levels and the differences between the elongated and shortened condi- tion, when stimulated by extremes of temperature, are not strik- ing to the eye. The additional fact that the cones, when maxi- mally shortened under the influence of temperature, never closely approach the external limiting membrane makes these animals rather unsatisfactory for certain kinds of experimental work. Tables 1 and 2 present data for both rods and cones from typical retinas at 5°C. and 25°C. in the dark. It will be seen that at the higher temperature the myoids of both cones and rods lengthen (figs. 32, 33). Especially in the cones is this response unmistakable. The lengths of the rod’s inner member, after subjection to the extreme temperatures, varies within only a few micra, yet the relative change may be 2.5 per cent or more; moreover, since the mean ranges at the extreme temperatures do not overlap, these differences are presumably significant. If the length of the rod ellipsoid, 4 p, MOVEMENTS IN THE VISUAL CELLS 153 TABLE 1 Measurements from the retinas offour Ameiurus which had been kept at 5'C. in the The values are in micra and represent measurements taken along axes dark. coinciding with radii of the eyeball NERVE 'IBER CHOROID TO NUMBER OF ROD INNER EXTERNAL LIMITING CONE MYOID ANIMAL EXT~~~~L~~ITING MEMBER MEMBRANE MEMBRANE 1 35 95 19-32 7-9 2 35 95 13-20 7-9 3 50 95 19-31 9 4 43 100 13-24 9-10 _- Mean ... . . 41 96 16-27 8-9 TABLE 2 Measurements jrom the retinas of four Ameiurus which had been kept at 85'C. in the dark. The values are in micra and represent measurements taken along axes coinciding with radii of the eyeball NERVE FIBER CHOROID To NUMBER OF LAYER TO ROD INNER BXTERNAL LIMIrING CONE MYOID hNIMA1. EXTERNAL LIMITIN( MEMBER MEMBRANE MEMBRANE 50 105 32-38 9-13 43 100 32-38 12-15 42 87 25-31 10 50 93 31--42 9-12 ___-_ - Mean 46 96 30-37 10-13 __ is subtracted from the mean values, the relation existing be- tween the corresponding values at either end of the temper- ature range will be expressed by the following proportion: 25°C: 5°C. = 3: 2. The variability in the length of adjacent rods in any section is relatively so great that this disparity in the length of the myoids is not apparent until actual measurements are made. In a few observations upon the effect of temperature on the extended rods in the light, the range at 25°C. seemed to run higher than at 5°C. by 15 to 20 per cent. The lengths of the extended inner members were approximately 50 p at 5°C. and 60 p at 25°C. Similar tendencies will be noted among certain other fishes. THE JOURNAL OF COMPARATIVE NEUROLOQY, VOL. 26. NO. 2 154 LESLIE B. AREY (2) Abramis. The cones, with their large oval ellipsoids 10 p in length, are very conspicuous in stained preparations. The fine rods, on the contrary, are not always demonstrable when treated with the Ehrlich-Biondi stain, which acts in an unusually capricious fashion in respect to these elements. The elongated cone cells are more or less uniformly extended, although the variability in length is greater under these con- ditions than when in the retracted state. The rods, however, are arranged in the dark at very irregular levels so that retinas present a fairly even distribution of them from 8 p to sometimes as far as 50 p from the external limiting membrane. In the light the condition is one of more uniform elongation. In tables 3, 4, and 5, are presented the data obtained from measurements at 5", 15", and 25°C. in the dark. TABLE 3 Measurements from the retinas OJ four Abramis which had been kept at kY2. in the dark; the values are in micra and represent measurements taken along axes coin- ciding with radii of the eyeball NERVE CHOROID TO ROD INNER NUMBER OP EXTERNAL LIMITINO EXTERNAL IIMITINP CONE MYOID MEMBER 1 (MAXIMUM) 1 ~~~~~~~E 1 MEMBRANE 1 1 90 90 7-12 2 72 85 5-12 3 85 91 5- 9 40 4 82 88 6-10 Mean.. . . . . . . . 82 89 6-11 40 TABLE 4 Measurements from the retinas of three Abramis which had been kept at 16°C. in the dark; the values are in micra and represent measurements taken along axes coin- ciding with radii of the eyeball NUMBER OF MEMBER ANIMAL (MAXIMUM) MEMBRANE 1 95 94 16-29 59 2 100 105 15-24 60 3 88 69 24-34 25 Mean. . . . . , . , . 1 94 I 89 I 18-29 1 48 MOVEMENTS IN THE VISUAL CELLS 155 TABLE 5 Measurements from retinas of four Abramis which had been kept at 25°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball CHOROID TO ROD INNEFI NUMBER OF LAYER TO EXTERNAL LIMITINQ CONE MYOID MEMBER ANIMAL EXTERNAL LIMITING MEMBRANE (MAXIMUM) MEMBRANE 1 80 89 2149 2 72 84 28-40 44 3 105 84 3&40 4 76 65 28-40 44 Mean. . . . . . . . . 83 81 27-42 44 The effect of temperature, therefore, upon the cones of Abramis is very marked, the length of the myoid at 5°C. (fig. 25) averag- ing only 25 per cent of that at 25°C. (fig. 27), while in extreme cases this ratio is as low as 10 per cent. If the mean limits of myoid extension are averaged, values of 9, 24, and 35 micra are obtained for the temperatures of 5", 15" and 25°C. respectively. 1.0: 2.5 : 4.0, or in other words, These values are in the ratio of the 'coefficient of expansion' for the myoid of Abramis is 2* for 10°C. ,4s a matter of fact, in the great majority of these temperature 5°C. represents a value too high and similarly 25°C. experiments, a value too low for the actual temperatures maintained. 3°C:. and 26°C. are more nearly the actual values. If temperature is plotted as abscissas and the myoid length in micra as ordinates, the resulting curve (fig. A) is a straight line showing that the temperature effect is uniform between these limits. The straight line obtained in the plot may indicate that the temperature response is the result of two or more opposed chemi- cal reactions which operate in a compensatory manner. Since in elongating is directly correlated the response of the myoid with the temperature gradient, it seems feasible that the effect of temperature is physical (in the sense of Herzog), and through its action chemical processes in the protoplasm are uniformly accelerated. If the length of the myoid is a fair index of the chemical activity that causes elongation, and if the effect of 156 LESLIE B. AREY temperature is purely physical, the coefficient of 2.; for 10°C. is of interest, on account of its agreement with the value found for the temperature coefficient of various vital processes as well as of ordinary chemical reactions. The incomplete data concerning the maximum lengths of the inner members of rods are hardly significant, although both of the mean values at 15°C. and 25°C. are slightlyabove the one measurement at 5°C. The Ehrlich-Biondi stain was used on most of these preparations and it was only rarely that the rod ellipsoid took the stain sufficiently to render its identi- fication certain. Big. A. length in the cone Table 6 gives measurements from, two retinas which had been to extreme temperatures in the light. The measure- subjected ments for the cone myoids are identical, and in no one of the four fishes was there a demonstrable change ascribable to temperature under these conditions. It should be noted that the cone measurements at 5°C. in the dark (table 3) are either equal to or, as in this case, are actually smaller than those repre- senting the highly retracted light condition. This dependence upon temperature was taken advantage of in all experimenta- tion to be described later where elongation of the cones was MOVEMENTS IN THE VISUAL CELLS 157 desired. Although the disparity between the rod lengths given in the table is probably extreme, such values have the same purport as the corresponding measurements made on the rods 'of Ameiurus . TABLE 6 Measurements from the retinas of two Abramis, one of which had been kept at 6"C., the other at 25°C. in the light; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball NERVE FIBER CHOROID LAYER ROD INNER NUMBER OF TEMPERATURE TO EXTERNAL CONE MYOID MEMBER ANIMAL "C. ToL~~~~~~ LIMITING (MAXIMUMI MEMBRANE MEMRRANE 1 5 2 25 , 88 54 1 1;: 8-14 8-14 I E (3) Fundulus. The retina of this fish is interesting because of the presence of prominent 'double cones.' Such elements are found in representatives of all the vertebrate classes, with the exception of mammals (Greeff, '00). They consist of two cones with fused inner members, although close examination is necessary to demonstrate this union. One component is usually larger and is known as the 'chief cone' (fig, 29; ell. con.), where- as the smaller is the 'accessory cone' (fig. 29; con. acc.) The chief cone alters its position independently of its accessory cone, which remains close to the external limiting membrane and is not moved to any great extent by the action of light or other stimulating agents. The rods, although rather small, are quite in evidence and differ from those of Abramis in maintaining fairly uniform de- grees of elongation. In the tables (7 and 8) which show the characteristic results of experimentation in the dark, the mean myoid length of the chief cones at 5°C. (fig. 28) is only about one-third that at the higher temperature (fig. 29). The differences in the accessory cones are not striking, although the lower values are somewhat increased at 25°C. Since the movements of the accessory cones are very limited, this probably represents a significant elongation. The contrast between the extension of the rod at 5" and 25°C. is striking and, added to the evidence gained from other fishes, 158 LESLIE B. AREY TABLE 7 Measurements from the retinas of Jive Fundulus which had been kept at 5°C. in the dark; the values are in micra and represent measurements taken along axes coin- ciding with radii of the eyeball NERVE FIBER CHOROID LAYER NUMBER OF To EXTERNAL CHIEF CONE ACCESSORY ROD INNER TO EXTERNlL ANIMAL MEMBER MYOID CONE MYOID LIMITING MEMBRANE MEMBRANE 100 69 5-1 1 1-5 16-19 106 68 1-5 88 69 5 1-5 105 68 3 1-4 11-19 75 52 5-9 1-5 16-23 Mean. ........ I 95 1 65 1 5-7 1-5 I 14-20 TABLE 8 Measurements from the retinas of Jive Fundulus which had been kept at 26°C. in the dark; the values are in micra and represent measurements taken along axes coin- ciding with radii of the eyeball NERVE FIBRE CEOROID LAYER NUMBER OF TO EXTERNAL CHIEF CONE ACCESSORY ROD INNER TO EXTERNAL ANIMAL LIMITING MYOID CONE MYOID MEMBER LIMITING MEMBRANE MEMBRANE 120 24 1 87 14-22 2-5 120 87 31 2 15-22 2-6 130 100 6 21 3 17-26 4 135 94 17-27 6 19 5 135 110 17-26 1-3 38 Mean ........ .I 128 96 16-25 3-5 27 indicates that in these animals a lengthening of the inner mem- bers is favored by a high temperature. A series of measurements of chief cones in the light failed to show any differences at the extreme temperatures. (4) Carassius. The retina of Carassius, as well as that of Fundulus, has prominent double cones. In the few eyes measured, the chief cone elongated with in creased temperature (figs. 23, 24) but the accessory cone did not change its position, at least to any extent. Table 9 summarizes the results from typical retinas. MOVEMENTS IN THE VISUAL CELLS 159 TABLE 9 Measurements from the retinas of four Carassius, two of which had been kept at 6"C., and two at 96°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball NERVE PIBER CaOROID LAYER NUMBER OF TEMPERATURE TO EXTERNAL TO EXTERNAL CHIEF CONE ACCESSORY ANIMAL "C. LIMITINCt 1 LIMITING 1 MYOID CONE MYOID MEMBRANE MmMBRANE 25 75 88 21 3-5 25 69 69 21 2-3 5 56 63 1-3 2 5 69 94 34 2-3 2. Frog. (1) Rana pipiens (adult). Gradenigro ('85)' Ange- lucci ('go), Hereog ('05) and Fujita ('11) have all stated that at a temperature of 30°C. or more, the cones of the frog shorten until they assume the position characteristic of light. Thus, 419) says: "Aufenthalt im Brutschrank 1/2 Stunde Hereog (p. lang, Temperatur von 21" bis 30" C. ansteigend: die- selbe zeigt auch, dass die Zapfen maximal contrahiert sind. Die Lange der Zapfen betragt mit ganz vereinzelten Ausnahmen 0.0091 mm. (v. d. Em. extern.-Oelkugel excl.) ." The action of low temperature was first tried by Herzog ('OS), who makes the following statement (p. 4241, concerning the re- sult of two hours' cooling to 0°C. in the dark: "Dagegen sind die Zapfen bereits nahezu horhstgradig verkurzt. Ihre Lange betragt fast durchweg 0.0078 bis 0.0091 mm." Fujita experimented upon six frogs, which were kept at a low temperature in an 'Eisgefass' for periods of 30 minutes 6 hours, after which the decapitated heads were fixed in to ice-cold fluid. His conclusion (p. 170) is diametrically opposed to that of Herzog. "Das Resultat war in allen Fallen das gleiche : ich konnte keine Helbtellung lconstatieren. Die Zap fen waren nicht kontrahiert. . . . . Measurements showing the elangation of the cone myoid at intermediate temperatures (14" to 19OC.) are not given by Herzog, 418) concerning retinas that had been who, however, states (p. raised in the course of 15 minutes from 18" to 24°C.: "Ein wesentlicher Einfluss is nicht zu erkennen. . . . . Die 160 LESLIE B. AREY Zapfenlange (von der Limitans externa bis zur Oelkugel im Ellip- soid exclusive) betragt im Durchschnitt maximal 0.034 mm., im Minimum 0.0169 mm." With apparatus and methods similar to those described in connection with the experimentation upon frog's retinal pigment, an attempt was made to discover the exact responses of the cone myoid to high, medium, and especially to low temperatures. In table 10, which gives the measurements of visual cells from a few typical preparations, it will be observed that the cones are greatly shortened at 33°C. (fig. 36), but that at the other temperatures they retain the elongated condition typical of darkness (figs. 34, 35).12 Measurements of the cones gave two modal lengths, which are approximately expressed by the figures in the table representing the extreme values. The maintenance of elongation at a low temperature is in agreement with Fujita's ('I 1) cxperiments,l? but is opposed to Herzog's conclusion, which was apparently based upon exhaustive investigation. It is true that the temperature of 0°C. which the latter worker used was a few degrees less than the lower limit (3" to 5°C.) employed TABLE 10 Measurements from the retinas of eighl Rma pipiens which had been kept at So, lao, 1g0, and SS'C., respectively, in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the egeball YERYE FIBER CHOROID LAYER NUMBER OF TEMPERATURE PO EXTERNAL CONE INNER ROD INNER '0 EXTERNAL ANIMAL 1 "C, LIMITING MEMBER MEMBER LIMITING MEMBRANE MEMBRANE 1 3 140 66 12-20 10 f 2 3 160 75 13-22 9* 11 * 3 14 103 56 11-20 88 11-16 10 * 4 14 50 103 13-19 13 * 5 19 61 19 94 63 14-23 10 =t 7 33 163 63 8-12 10 * 8 33 126 63 7-10 11 =I= 1* The actual values at medium and high temperatures vary somewhat from those given by Herzog for R. temporaria (and R. esculenta?). l3 This experimentation upon the retinal elements of the frog was practically completed before Fujita's paper was known to me. MOVEMENTS IN THE VISUAL CELLS 161 by me, yet it seems improbable that such a small difference would cause the cones to shorten maximally. Any error that could be introduced during these determinations would tend to shorten the cones, hence Herzog's results are at a disadvantage in this respect. I have continued experiments for 6 hours, yet the results were always the same; in no case was there found a gen- eral shortening of the cones that in any way resembled the ex- treme condition at 33°C. From these results, which, in a general way, are the reverse of those found in fishes, it is evident that the responses of the cones are not comparable to those of the retinal pigment. The so that stimulat- pigment may indeed be under nervous control, ing agents such as heat, cold and light produce a migration ac- cording to the principle of specific energies, yet if the cone cells are influenced by the nervous system, these experiments can not be said to furnish proof of such a relation. In order that there should be no doubt concerning the effect of low temperature upon the cone myoid, a further determina- tion was made long after the results which are tabulated above were obtained. In this later experiment rigorous precautions were observed to eliminate possible errors. After frogs (R. pipiens of various sizes), kept at a temperature of 18"C., had been subjected to a preliminary treatment of darkness for 72 hours, they were introduced into a vessel cooled to l"C., where they remained for a period of 4 hours. The eyes were then quickly excised in dim, red light, the operation not requiring more than 15 seconds, after which they were returned into darkness where fixation at approximately the freezing temperature ensued. The average measurements of the cone myoids in these prepara- tions were as follows: 12 to 14 p; 10 to 15.4 p; 10 to 14 p; 10 to 11 p; 13 to 15.4 p; 9 p. These values, although somewhat smaller 10, can not be said to prove that low than those given in table temperature shortens the myoids as does high temperature. Gradenigro ('85) found that elevated temperature induced a shortening of the rod in the dark. After measuring the myoid length in a considerable number of preparations from retinas subjected to various temperatures both in light and in darkness, 162 LESLIE B. AREY I was unable to discover constant differences in length that could be correlated with definite temperatures. The agreement of mean values obtained from various retinas under identical temperature conditions was not good, and since the rod myoid 6 p to 12 p in length, even small variations fur- measures only nish serious obstacles in determinations of this kind. In any one preparation, moreover, variability in the length of adjacent myoids tended somewhat to mask a possible temperature effect. If anything, my measurements showed the reverse of what Gradenigro maintained, the elongation at 33°C. in the dark being greater than at 5"C., but, as stated before, these results are by no means trustworthy. A shortening of the rod through the action of high tempera- ture, as claimed by Gradenigro, is of interest because, accord- ing to most investigators, light produces the same result. With this can be compared an analogous correlation in fishes, where light causes an elongation of the rod myoid and, as I have shown, elevated temperature does likewise. It is certain, on the con- trary, that although the cones of both frogs and fishes shorten in the light, heating produces unlike responses in the dark. (2) Rana catesbjana (larvae). Although the cones in both the 4.5 em. and the 7.0 em. larva of this frog are of large size, clearly defined temperature responses were not observed ; in- deed, the difference between the positions assumed even in light and darkness is not striking, the cone myoids in the light re- maining well elongated in comparison to those of the adult R. pipiens. The variability in length in different preparations is considerable, yet if anything, the cones appeared more shortened at 3°C. than at higher temperatures. There is no marked shorten- ing at 33"C., for the cones under these conditions were as long as at lower temperatures, and in some cases longer. It is probable that the responses of the cones in adult R. catesbiana will be found to agree with those in other species which have been studied, although no experimentation was performed to deter- mine this point. 3. Necturus. The rods, and the single and double cones of Nec- turus are very large, yet positional changes with varying tempera- MOVEMENTS IN THE VISUAL CELLS 163 ture (3" to 28°C.) were not observed. Individual cells vary more or less in the height at which they are situated above the exter- nal limiting membrane, yet no constant differences of significant amount could be correlated with definite temperatures. With these results should be compared Garten's ('07) denial of a change in the position of the cones of the 'salamander' through the influence of light, such as Angelucci ('90) had previously claimed. Stort ('87), however, described movements in both the rods and the cones of Triton. C. EXPERIMENTATION UPON EXCISED EYES a. E$ect of light and darkness The results of a number of investigators since the first work of Englemann ('85) have indicated that the retinal elements of some vertebrates, and especially the frog, are subject to a nervous control, the action of which is not well understood. The r81e which the nervous system plays either in producing or in assisting the movements of the various retinal elements is hard to demonstrate. Experimentation involving the direct action of light on excised eyes can not be expected to solve the problem decisively, for if no movements result, autoanaestheti- zation, or some similar disturbance due to the interrupted blood supply, may be the real cause. If, on the other hand, responses are called forth by direct stimulation, it by no means follows that a similar phenomenon necessarily occurs in the living animal, any more than a demonstration of the direct stimulation of muscle fibers proves that this rather than a nervous impulse is the normal method of muscle stimulation. The limitations which restrict a wide interpretation of results, however, do not lessen the interest involved in determining the extent to which the retinal elements can be directly stimulated. Hamburger ('89) maintained that the cones and retinal pig- ment in excised eyes of the frog assumed the positions characteris- tic of light or darkness according to the conditions of the experi- ment. Dittler ('07) working on isolated frog's retinas obtained 164 LESLIE B. AREY a shortening of the cones in localized areas through the action of light but found darkness to be ineffectual. The spread of the response to portions of the retina unstimulated by light, led Dittler to investigate further the cause of cone retraction. He was able to furnish experimental proof that weak acids, resulting from catabolic processes in the retina, caused the cone myoid to shorten; hence he concluded that the cone myoid was not of itself ‘lichtempfindlich,’ as Englemann (’85) had believed, but was stimulated to movement through chemical agents result- ing from the action of light on the retina. Fujita (’ll), as a result of very limited experimentation, stated that the pigment of the excised eye of a frog expanded in the light but did not con- tract in the dark. Ringer’s solution, normal saline solution, and tap water were used by me for the immersion of excised eyes. When the first two media were employed the movements of the rods and cones of Ameiurus, through the action of light, were never clearly are to be interpreted as demonstrated; possibly such results evidence of a chemical control somewhat comparable to that de- scribed by Spaeth (’13) for the melanophores of Fundulus. Tap water did not inhibit the movements of any of the retinal ele- ments of Ameiurus and consequently it was used in all subse- quent experimentation. The pigment of Ameiurus, Abramis and Fundulus did not contract when excised eyes from light-adapted fishes were sub- jected to darkness for periods of 4 hours or less. At most there was only evidence of a retraction of the distal accumulation of pigment, which is characteristic of light-adapted eyes, to form a more homogeneously pigmented zone (figs. 2, 10, 6). When the reverse experiment (subjection to light) was performed, the pigment of Ameiurus became maximally expanded in 2 hours (figs. 1, 3). Only the slightest tendency toward expansion, however, could be found after similar experimentation on the two other fishes. It thus appears that light acts directly on the pigment of Ameiurus only, while darkness is totally ineffec- tive on all three animals. MOVEMENTS IN THE VISUAL CELLS 165 When the rods and cones of Ameiurus and the cones only of Abramis and Fundulus were tested, the following results were obtained. The rods of Ameiurus moved both in light and in darkness, whereas the cones were stimulated only by light, no elongation occurring in the dark even when the experiment continued for 4 hours. The cones of Abramis and Fundulus did not change their positions to any extent either in light or in darkness. In the light the cones of Abramis, which were more carefully investigated than those of Fundulus, at most showed only slight retraction and never closely approached the exter- nal limiting membrane. If light did exert a direct influence on the cones or retinal pigment of this fish, the changes would be extremely easy to distinguish due to the wide difference between the light- and dark-adapted phases. Since neither the cone cells nor retinal pigment of Abramis underwent movements under these conditions, it is possible that the accumulation of catabolic products, occasioned by the interruption of the blood supply, was responsible. Experimenta- tion of the following kind shows the importance of maintaining the vascular circulation. If the optic nerve only of Abramis is cut, the retinal elements undergo their normal movements in darkness and in light. If, however, all the blood vessels and muscles are cut and the eye ball is attached to the body by the optic nerve only, no movements result. The objection may be made that some nervous mechanism is deranged by cutting these muscles and blood vessels, but this is hardly probable, as further experimentation, to be presented in a subsequent paper, on this and other fishes has shown. In cases like the movement? of the pigment or cones in excised eyes of hmeiurus through the action of light only, it is probable that an inhibitory tendency is also present, but the response to the stimulus furnished by light is sufficiently vigorous to over- come it. Either a less vigorous stimulus or response niay ex- plain why no movements of the cones and pigment occur in darkness. An inhibition due to the presence of unremoved catabolic products, as postulated here, would be merely a form of auto- 166 LESLIE B. AREY anaesthesia. As will be shown, carbon dioxide and other anaes- thetics do, in fact, arrest the movements of all the retinal elements of fishes. Dittler (’07) accounted differently for the absences of elonga- tion in the cones of isolated frog7s retinas which were introduced from light into darkness. In order to appreciate his way of viewing this situation, it is necessary to understand the general theory advanced by him to explain the movements of the cones. In darkness an equilibrium was supposed to exist in the metab- olism of the retina, the elongated cone myoid representing an unstimulated condition. Through the action of light, however, catabolic processes preponderate, and the accumulated acid wastes chemically stimulate the myoid to shorten. These con- clusions were based upon experimental evidence by which it was shown that weak, free acids could be detected if isolated retinas were subjected to light in limited amounts of Ringer’s solution, and further that such an acid solution was capable of causing other dark-adapted cones to shorten while still in the dark. To return to the case under consideration, Dittler believed that the accumulation of the catabolic products formed in the light merely continued its contractile influence after the isolated retina was removed into the dark, and since these products were not removed, the metabolic equilibrium could never be restored and consequently elongation failed to occur. This theory of chemical stimulation is not supported by the condition in Fundulus and especially in Abramis, where the cones of excised eyes do not shorten even when exposed to light, for under these favorable conditions the tendency toward the production of a catabolic excess should be maximum. In still another way Dittler’s theory does not explain a typical response of the cones of fishes. The cone myoid in isolated retinas of the frog shortens when the temperature is raised to 30°C. or more in the dark (fig. 36), and this fact Dittler used to sup- port his view in the following logical manner. It is well known that most chemical reactions are accelerated by raising the temperature; hence in this case the autonomic equilibrium normally existing in the dark would become disturbed, the re- MOVEMENTS IN THE VISUAL CELLS sulting increase of catabolic products causing the cone myoid to shorten.l* Although Dittler’s statement is not altogether clear, it seems evident that low temperature was supposed not only to reduce the metabolism of the retina to a low level, .but also to render the cone myoid less ‘empfindlich’ to chemical stimulation. The conditions in the dark-adapted cones of fishes, however, are entirely different, for here not only do the cone myoids elongate when the temperature is increased, but also elongated cones can be made to shorten by the use of low temperatures. If the shortening of the cones of fishes in the light were due to a chemical stimulation, how can the elongation of these elements in the dark through the action of heat be explained, since mani- festly in this case the metabolic equilibrium tends to become destroyed, the result being the formation of an excess of catabolic wastes, which by analogy with the conditions in the frog, should cause the cone myoids to shorten? Moreover, the efficiency of low temperature in retracting elongated cones, and the cor- relation between the uniform degree of myoid elongation and the temperature gradient (p. 155) finds no explanation through Dittler’s hypothesis. It should be said, however, that Dittler strictly limited his conclusions, the experimental evidence for which appears to be well established, to the material upon which he worked, and was even reserved in suggesting the occurrence of a similar method of stimulation in living animals. The reason why the rods of Ameiurus move in darkness, while the cones do not, may be as follows. The rod myoid normally shortens in the dark, whereas the cone myoid elongates. It is probable that the contractile function of the myoid is more vigorous than the reverse process of elongation. This is not only substantiated by the fact that dark adaption of the rod takes less time than light adaption, but also by experiments 14 Dittler did not formulate this conception in extenso as I have expressed it, yet several statements (pp. 317-318) show that this was his belief. A concluding quotation reads: . . . . “der Einfluss der Temperatur uberhaupt ganz nach physikalischen Modus zu Wirken scheint, und berechtigt uns, seine Wirkung rein in diesem Sinne zii fassen.” 168 LESLIE B. AREY in which the optic nerve was cut. In such cases the rod never elongated in the light although it often showed a tendency tJo shorten when introduced into the dark, whereas t,he behavior of the cone cells in light and darkness was the exact opposite to that of the rod, since they tended to shorten in the light but remained unchanged in the dark. On these grounds, therefore, an explanation is offered to show why it is that through the strong stimulation produced by the direct action of light, both types of cells show characteristic responses, while in the dark only the more vigorous contractility of the rod myoid becomes effec- It must be remembered, however, that although the direc- tive. tion of the movements of the rod and cone cells are opposed, the real response of the protoplasmic myoid may be similar in If this were true, the apparent inconsistency in both cases. the movements of these elements would be due to a difference in the axis of contractility in the two kinds of myoids, and the explanation just advanced would not stand. Reference will be made to these possibilities in another place. b. E*fect of temperature Previous attempts to determine the direct influence of tem- perature upon the retinal elements have been confined to the frog. Gradenigro ('85) found that if excised eyes of dark-adapted animals were subjected to a temperature of 30" to 36°C. the rods and cones shortened and the pigment expanded, both end-results being characteristic of light-adaption. Dittler ('07) was able to confirm Gradenigro's discovery concerning the cone cells. When the isolated retina was heated to 35" to 37°C:. in the dark for 50 to 60 minutes, the cone myoids shortened. After retinas had been subjected to a temperature of 1" to 2°C. for many hours, on the contrary, no shortening of the cone myoid was observed. The apparatus and methods used by me were similar to those described in connection with the experiments upon living fishes. The excised eyes were contained in test tubes which were sus- pended in jars of water kept at appropriate temperatures. Eyes MOVEMENTS IN THE VISUAL CELLS 169 from the same animal were used simultaneously, one at each temperature extreme. 1. E$ect of temperature upon retinal pigment. After many trials it was found that sharp differentiation of the retinal pig- ment of fishes was hard to secure at the extreme temperatures when the initial temperature had been intermediate. Accord- ingly, the expedient was employed of subjecting the living ani- mals to a preliminary treatment either at 3°C. or at 25"C., and as a result uniformly satisfactory differentiation was obtained. The retinal pigment of Ameiurus behaved precisely as in living animals. At a low temperature, both in darkness and in light (figs. 3, l), the degree of distal migration was greater than that at a high temperature (figs. 4, 2). Particularly in experiments conducted in the light was this strikingly appar- ent, since in many preparations at 3°C. the pigment migrated so far distally that the more proximal portions of the cells were free of granules, while a sharp line of demarcation existed be- tween the pigmented and non-pigmented zones. A few experiments were made upon the dark-adapted eyes of Abramis. In this case also, a greater pigment expansion was found at 3°C. than at 25°C. (figs. 11, 12). Reference has been made to the contention of many investi- gators that there is an apparent nervous control over the move- ments of the frog's retinal elements. It has been shown that at a medium temperature in the dark the pigment is maximally contracted (fig. IS), whereas at higher and lower temperatures (figs. 17, 19) a considerable degree of expansion is effected. Not only is the amount of migration occasioned by temperature much more extensive than that in fishes, but also the similarity between the effects of the two temperature extremes as con- trasted with an intermediate temperature, has no parallel among other pigment cells or even in melanophores. Since it is at least agreed that the pigment of excised eyes of the frog expands in the light, it ought to be possible to observe the effect of temperature, if this agent acts directly upon the pigment cells. Excised eyes from animals that previously had been at an intermediate temperature in the dark, were subjected TEE JOURNAL OF COMPARATIVE NEUROLOQY, YOL. 26, NO. 2 170 LESLIE B. AREY to temperatures of 3", 16", and 33"C., but no changes were ob- served in the position of the retinal pigment. These results, which do not agree with Gradenigro's statement, by no means furnish conclusive proof that in the living frog temperature operates through the nervous system, yet when supported by a comparative study of the retinal pigment and melanophores of other animals, the conclusion reached by Hereog ('05), that the expansion of the frog's retinal pigment under these circumstances is of nervous origin, involving the principle of specific energies, becomes highly probable. According to Fujita ('ll), when excised eyes from dark-adapted frogs are retained in the dark for 20 minutes, the pigment assumes a partial light position. My observations do not confirm this result, for no migration of any consequence occurred. 2. E$ect of temperature upon visual cells. When a study of the visual cells of dmeiurus was made, the effect of temperature was found to be essentially similar to that upon normal animals. When eyes from dark-adapted individuals of Abramis that had previously been kept at a temperature of 25°C. were like- wise excised and subjected to 5°C. and 25°C. in the dark, those at 25°C. (fig. 27) retained the elongated position while those at 5°C. (fig. 25) shortened to a considerable extent although somewhat less than in living animals. Mention has already been made of the significance of these results in con- nection with the applicability of Dittler's theory of chemical stimulation to the cones of fishes. Although the rods of Abramis at both temperatures showed a distribution extending over wide limits, yet the shortest measured 12 p at 5°C. as compared with 20 p at 25"C:., and the modal elongation at the same temperatures, as judged by the eye, was 18 p and 25 p respectively. It is interesting to note that in the excised eyes of dark-adapted Abramis, temperature is able to produce changes in both the retinal pigment and visual cells, notwithstanding the fact that light and darkness are wholly ineffectual in this respect. Table 11 summarizes these results from typical retinas. A few experiments were performed upon the cone cells of The results obtained were identical with those stated the frog. MOVEMENTS IN THE VISUAL CELLS 171 TABLE 11 Measurements of the visual cells from the retinas oj two Ameiurus and two Abramis, of which one of each had been kept at 6°C. and the other at 96°C. in the dark; the values are in micra and represent measurements taken along axes coinciding with radii of the eyeball FISH I TEMPERATURE "c. 1 CONE MYOID ROD INNER MEMBER Ameiurus No. I... ..... 4-16 5-6 Ameiurus No. 2... ..... 8-12 -1 2: 5 19- 32 Abramis No. l... ...... 10-30 18 Abramis No. 2... ...... 35-50 25 I 25 ___-__ by Dittler ('071, who used isolated retinas. In dark-adapted eyes which were placed in water at a temperature of 33°C. the cone myoids shortened (fig. 36), while at 3°C. or 16°C. (figs. 34, 35) the myoids remained for the most part unchanged. These re- sults are identical with those found by Fujita ('11) and myself on the cones of living animals, and indicate that, unlike the pig- ment cells, the niovements of the cones are not dependent upon nervous control. If an influence of the nervous system over these elements exists in the normal animal, it is at least not mani- fested as is the control over the retinal pigment, in which changes at both high and low temperatures can be interpreted according to the principle of specific energies. D. EFFECT OF ANAESTHETICS Various instances have been noted throughout this paper in which the behavior of the retinal pigment and the visual cells, when deprived of t,heir blood supply, cast suspicion upon auto- anaesthetization as being the factor causing suspension of move- ment. Certain conditions discovered in the responses of melano- phores in the web of the frog's foot had previously suggested such a possibility; indeed, it was this difficulty which led to the abandonment of the frog's melanophore as material for an investigation somewhat similar to the present one. In this way my interest was aroused to determine the effect of anaesthetics on the movements of the retinal elements, both in normal animals and through the more direct action upon excised eyes. 172 LESLIE B. AREY The effect of certain drugs, as quinine and strychnine, upon the retinal pigment (‘protoplasmagifte’) is in dispute. It is clear, however, from the work of Ovio (’95) and of Lodato (’95) that cocaine can arrest pigment migration. As a precaution against a possible source of error, animals were never introduced from one condition of light or darkness to the other without having been previously subjected to a brief preliminary treatment of the anaesthetic which was to be tested. a. Retinal pigment I. Carbon dioxide. The carbon dioxide used in these experi- ments was a commercial soda-water product sold under the trade name of ‘Pureoxia.’ Quantitative determinations of the con- centrations used were made by titration with & sodium carbonate, using phenolphthalein as an indicator. In the first experiments made on Ameiurus the movement of the pigment was arrested by a strong solution of carbon dioxide, but since none of the animals survived such treatment the obvious objection exists that the pigment cells also may have been killed. A slight refinement in method consisted of revivifying the fishes at intervals, by temporary removal to running water, until opercular movements were restored. By this method fishes were kept alive for 2 hours, during which time four or five revivifying treatments were necessary. The migration of retinal pigment was shown to be checked both in light and in darkness, yet controls proved that the cells were not permanently injured. A method which gave more satisfactory results was devised after repeated trials had given a mixture of tap water and car- bonated water of sufficient strength to anaesthetiee an Ameiurus but not to prohibit opercular movements of greatly reduced amplitude. The record of an experiment will well illustrate both the method and the results. Experiment 4.1 6. A dark-adapted Ameiurus was placed in a mixture of 1 part of ‘Pureoxia’ to 4 parts of tap water, and after re- MOVEMENTS IN THE VISUAL CELLS maining 10 minutes in the dark the jar was removed into strong dif- fuse daylight for la hours.. During this time, the fish was practically motionless except for a very weak but rhythmical pulsation of the oper- cular rims. At the end of the experiment one eye was removed and fixed. The Ameiurus was allowed to recover until the next day when the other eye was removed. The pigment in the eye which had been subjected to carbon dioxide was in the typical dark position (cf. fig. 4) while the pigment of the control eye was maximally expanded (cf. fig. 2). Titration of the anaesthetizing solution showed that the concentration of carbon dioxide had been in the ratio of 60.14 cc. per litre of water. In the converse experiment from light to darkness an Ameiurus lived 3 hours in a similar solution (53.07 cc. of carbon dioxide per litre) during which time the pigment retained its light dis- tribution, whereas the control eye removed on the next day, showed maximal contraction. These results prove conclusively that in the presence of certain concentrations of carbon dioxide the pigment cells are not injured but are in a condition of anaesthetization whereby there is a failure to respond to the normal stimulus causing contraction and expansion. Such experimentation, however, does not show whether this failure is due to a direct effect upon the pigment cells or to an inhibition through the central nervous system. To demonstrate which alternative is true, the effect of carbon dioxide was tested on excised eyes of Ameiurus. If; under these conditions, a migration occurs a direct influence of the anaesthetic on the cell itself will be disproven, while on the other hand, if no migration ensues one can only infer that a similar direct action on the pigment cell is responsible for the whole course of events in the living fish, whereas an inhibition through the cen- tral nervous system may be involved as well. For such an experiment the excised eye of Ameiurus is well adapted, since its pigment has been shown to migrate from the dark to the light position, although the reverse process does not occur. Excised eyes of dark-adapted fish were exposed to light in a solution of carbon dioxide having a strength of about 60 cc. per litre. The pigment in each case was arrested in the contracted position. 174 LESLIE B. AREY The work described for living Ameiurus has been repeated on both Abramis and Fundulus with identical results. In every case the pigment maintained the position it occupied previous to the application of the anaesthetic. The results obtained in this study, as a whole, are very differ- ent from those of Fick ('go), who concluded that the retinal pig- ment of the frog expanded when the animals were subjected to an atmosphere of carbon dioxide gas. Fick attributed this result to asphyxiation and it is certain that the experimental conditions in his work differed greatly from those in my tests. In order to make the experiments more comparable, frogs should be treated with a mixture of oxygen and carbon dioxide gases in which they could live. 2. Ether. The anaesthetic effect of ether on the retinal pig- ment was demonstrated by a series of tests that duplicate those described with carbon dioxide. Care must be observed against using an excess of ether since otherwise a partial or complete disintegration of the pigment cells results. Both dark and light trials were made on Ameiurus, Abramis, and Fundulus. In each animal the pigment was found to be completely arrested in whatever position it occupied at the be- ginning of the experiment. Controls proved that ether, if used in small amounts, does not permanently injure the pigment cells. Ether also checked the migration of pigment in excised eyes of dark-adapted Ameiurus when such eyes were subjected to light. 3. Chloretone and urethane. These substances are such satis- factory narcotizing agents that their effect was tested upon the retinal pigment of Ameiurus. Individuals lived in 0.1 per cent chloretone or in 1.0 per cent urethane, but the pigment was not arrested in its movement from the dark to the light phase. In concentrations of 0.5 per cent chloretone and 2.5 per cent ure- thane, the pigment migrated when fish were brought from dark- ness to light although the animals died in both cases. The results from all the foregoing experimentation are of interest in showing the difference in the effect upon pigment cells of four powerful anaesthetics, of which only two were MOVEMENTS IN THE VISUAL CELLS 175 efficient. The experiments with chloretone and urethane also prove that even though the animal as an organism dies, the pigment, nevertheless, can expand independently. b. Visual cells Experiments similar to those just described were repeated in order to determine the action of anaesthetics on both rod and cone cells. Since the cone myoid is maximally elongated at about 25°C. in the dark (figs. 25, 27), this condition was taken advantage of in producing sharp contrasts between dark and light phases. The cones of Abramis and Fundulus, on account of their great contractility, were particularly favorable for ob- servation, as were the rods of Ameiurus because of their large size. The results of these experiments are shown in table 12. TABLE 12 A tabulation of the effects of carbon dioxide and ether upon the rrLovernents of the visual cells of Ameiurus, Abramis, and Fundulus; X indicates that the movements of the elements were completely arrested; conditions corresponding to the blank spaces were not investigated _____ CONE ROD FISH 1 Dark to Light 1 Light to Dark- 1 Dark to Light I Light to Dark Ameiurus . . . . . . . X Abramis . . . . , . . . X x x Fundulus . . . . . . . x x The conclusion is, therefore, that both ether and carbon diox- ide anaesthetize the visual cells of normal fishes to such an extent that neither light nor temperature is effective in causing positional changes. A few experiments upon the excised eyes of Ameiurus showed that both-carbon dioxide and ether have the same anaesthetic effect on the rods and cones as that described for normal animals. Whether or not autoanaesthetization prevented movements of the retinal elements, as was suspected in previously described experiments when the normal blood supply was interrupted, it is at least demonstrable that certain anaesthetics do act in et 176 LESLIE B. AREY similar way. The effect of carbon dioxide is especially interesting for, as it is the commonest catabolic product, it may have been the agent that prevented movements in those cases. This con- ('07) view, which assumes the ception is opposed to Dittler's existence of a balance in the metabolism of the unstimulated cone cells which is disturbed by the increased catabolism through the action of light. The movement of the cone cells in the isolated retina of the frog was stated by Dittler to be due to the action of a weak free acid, the product of increased catabolism. This conclusion, which was supported by experimental evidence, is opposed to that postulated by me; nevertheless, it must be pointed out that the responses of the retinal elements differ considerably in fishes and in the frog, and while evidence for autoanaesthetization is indirect yet the results obtained from experimentation upon fishes can be consistently interpreted in this way, whereas Dittler's hypothesis does not meet all the known facts. A discussion of these points was given in anot*her section of this paper. E. EFFECT OF OXYGEN Spaeth ('13) showed that the isolated melanophores of Fundu- lus contract in the absence of oxygen, but contracted melano- phores do not expand when oxygen is the only stimulating agent present. Fick ('90) deprived dark-adapted frogs of oxygen by submergence in water or by introducing them into an atmosphere of hydrogen or carbon dioxide. As a result of this treatment he asserts that the retinal pigment underwent expansion. Dittler ('07) states that in frogs which are about to hibernate the cones are never as fully elongated as in active animals; but after subjection to an atmosphere of pure oxygen the cones can again be obtained in the maximal dark position. It seems probable that in this case the effect of okygen was indirect, and the increased activity of the cone cells accompanied pari passu the return of other body activities. In order to test whether or not the amount of oxygen avail- able to a fish in any way controls the distribution of its retinal MOVEMENTS IN THE VISUAL CELLS pigment, a series of experiments, chiefly upon Ameiurus, were performed. In experiments involving a reduced oxygen supply, the ap- paratus was simple. A 33 litre flask was filled with boiling water. The flask was then closed with a three-hole rubber (1) a glass tube extending to stopper through which passed, the bottom of the flask, which served to introduce gas from a hydrogen generator, (2) a glass overflow tube extending about three-quarters of the way down the flask, which served chiefly as an outlet for the hydrogen gas, (3) a mercury pressure regu- lator. As soon as the flask of boiling water was stoppered, the hydrogen supply was turned on and as a result, water was forced to escape through the overflow tube, its place being taken by hydrogen gas. When the water level reached the bottom of the overflow tube no more escaped, but the gas after bubbling through the water did do so and was conducted through a water trap to the outside air. As the water cooled down to room temperature it could not take up oxygen since none was present, and furthermore, the bubbling hydrogen gas tended to expel mechanically any residual oxygen present in the boiled water. Water containing an excess of oxygen was prepared by bub- bling oxygen gas through water in a flask similar to that de- scribed in the former experiment, whence it escaped by means of an overflow tube leading into a water trap. The water used had previously been boiled and reoxygenated by an aquarium aerating device . Quantitative determinations of the oxygen content were made at the expiration of all experiments by the method of Winkle (Treadwell and Hall, '05). Ameiurus was used in most of the experimentation, although hbramis served in a few cases. The description which follows applies particularly to Ameiurus. It was possible to reduce the oxygen supply to an amount in which the fish could not live.15 This, for example, happened l5 The normal oxygen saturation of water at 20°C. is 6.356 ee. per liter (Tread- well and Hall, '05). was found Boiled water which had been cooled rapidly to contain about 0.93 cc. per liter. 178 LESLIE B. AREY when only 0.8 cc. of oxygen per litre was present. On the other hand, in water containing an excess of oxygen (7.5 cc. per litre) respiratory movements of the operculum ceased, the fins appeared reddish in color and respiration may have been largely cutaneous. In parallel experiments conducted both in the dark and at various light intensities, no difference could be detected in the positions of the pigment or visual cells under the extreme conditions of oxygen supply. This is not surprising, for pre- sumably but little oxygen is needed to permit the cells to func- tion, and since for the success of the experiment, the animal must have enough oxygen with which to keep itself alive, a crucial test involving a complete elimination of oxygen is not possible. Since t’he pigment can not be made to contract in excised eyes, but only to expand, a decisive experiment in which all oxygen might in this way be eliminated (similar to Spaeth’s work on isolated chromatophores) was impossible. Pigment, rods, and cones respond in a normal fashion when brought from darkness to light or vice versa in water contain- 0.9-1.0 cc. per litre, ing the minimum oxygen content, about in which the Ameiurus can live. Since no indication was observed of a tendency toward ex- pansion in the retinal pigment cells of fishes which were deprived of oxygen, it is evident that the expansion described by Fick (’90) in dark-adapted frogs whose respiration rate had been re- duced by covering the head with a velvet hood, is exceptional. In view of the well known respiratory function of the frog’s skin it is possible that Fick’s results are open to other interpretations, especially since his experiment, a repetition of the earlier work of Englemann (’85), is not in agreement with the latter’s con- clusion relative to the absence of movement in the retinal ele- ments when frogs provided with velvet hoods were retained in the dark as controls to other experiments. The chief value, therefore, of the work done by me is to show that within normal experimental limits the retinal pigment and visual cells of fishes are not affected by an increased or diminished oxygen supply. MOVEMENTS IN THE VISUAT, CELLS 179 F. INTERRELATION OF INTEGUMENTARY PHOTO-RECEPTORS AND RETINAL ELEMENTS The skin of several lower vertebrates has been shown to be sensitive to light. Among the fishes, Eigenmann ('00) stated that certain blind forms living in caves gave motor responses when stimulated by light, the photo-receptors presumably being located in the skin. Parker ('05) followed up some negative results obtained by one of his students on Fundulus by an investi- gation on ammocoetes, and proved that the integumentary nerves were sensitive to light, causing movements of the animal that were both 'phototropic and photodynamic.' A photo- receptivity of the skin of certain other vertebrates was first demonstrated by the following workers: Graber ('84) on Triton; Dubois ('90) on Proteus; Korcinyi ('92) on the frog; Carleton ('03) on Anolis; and Eycleshymer ('08) on Necturus. Englemann ('85) covered the heads of dark-adapted frogs with a velvet cap and exposed the bodies to sunlight. Under these conditions, he asserted that in 15 minutes the pigment and cone cells assumed the maximal light position, whereas the same elements in control experiments conducted in the dark remained unchanged. Illumination of the skin for longer periods a falling off ('herabsteigen') in the expansion was said to result in of the retinal pigment and to a weakened response on the part of the cones. From these results he concluded (p. 507) : ". . dass Zapfen und Pigment des Auges von entfernten Korpergegenden aus reflectorisch in Bewegung gebracht werden konnen." Fick ('90)' in repeating this experiment of Englemann, found the pigment in an expanded condition while the frogs, ready for the test, were still in the dark, and after supplementary experimentation of various kinds he decided that pigment ex- pansion accompanied disturbed respiration. In the case under consideration the velvet hood was supposed to have caused partial asphyxiation. KoriLnyi ('92) refers to the similarity in the responses of the retinal pigment resulting either from the illumination of the retina or of the skin only, yet he does not state that he actually observed this condition himself. 180 LESLIE B. AREY More recently Fujita (’11) asserted that after the head and forward extremities were bound with wet black cloth and the rest of the body was exposed to sunlight for 15 to 20 minutes, the ‘eyes’ remained in the dark-adapted condition. I, myself, before learning of Fujita’s work, had performed several experiments of the same kind. The head and fore body of dark-adapted frogs were bandaged with many thicknesses of black velvet and the remainder of the body and the hind legs were exposed to direct or diffuse sunlight for periods of 15 minutes to 1 hour. Care was taken to keep the skin moist and to guard against the heating tendency of direct sunlight by using a heat filter. Although the animals were in an active condition at the end of the experiment, there was in no case a distinct change in the position of the cone cells or retinal pigment as Englemann maintained. S. G. Wright, no In an unpublished investigation by Mr. direct responses were observed when Ameiurus, from which the eyes had been removed, were illuminated with the light of an electric arc. The normal fish, as is well known, is a night feeder, yet it also frequented equally the light and dark halves of an aquarium jar. It is, conceivable, however, that the soft skin of this animal contains a photoreceptive mechanism, even though the motor responses to light fail to indicate its presence. One of the pos- sible ways in which an integumentary photosensitivity could be manifested is through an influence on the position of the retinal elements, similar to the relation which Englemann be- lieved to exist in the frog. In view of such a possibility several experiments were performed in which dark-adapted fish with bandaged heads were exposed to daylight and to the light of an electric arc for periods of 45 minutes to 1 hour. In no case, however, was there the slightest tendency toward movement on the part of the rods, cones, or retinal pigment. These results indicate that neither in the frog nor in Ameiurus are movements of the retinal pigment or visual cells evoked a cooperation of dermal photosensitivity and ‘retino-motor, by nerve fibers. Consequently, Englemann’s experiment does not MOVEMENTS IN THE VISUAL CELLS 181 have the significance that he believed it possessed, when he attempted to furnish physiological proof that in the frog: “Jeden- falls aber laufen . . . . auch retinomotorische Fasern von den grossen Nervencentren aus durch den Sehnerv zurn Auge” (’85, p. 506). 4. DISCUSSION We have so accustomed ourselves to view the phenomena exhibited by living organisms from the evolutionary standpoint that an ‘explanation’ which will reveal the adaptiveness of an organism to its environment is demanded whenever a system of relations involving constant responses to definite stimulating agents is discovered. To make dogmatic assertions regarding the presence or ab- sence of adaptation in a set of responses is, obviously, a matter of exceeding danger, yet if the phenomena exhibited by a series of representative animals to definite stimulating agents are shown to be variable, it is at least evident that a single inclusive explanation will not be forthcoming. The writer has attempted to show elsewhere (hey ’15) that the discontinuous occurrence of photomechanical responses in the visual cells and retinal pigment, both in the various verte- brate classes and among different representatives of certain individual classes, renders it extremely difficult from the adapta- tional standpoint to devise a satisfactory explanation for the meaning of these movements. The maiority of theories which attempt to link the known responses of the retinal ele- ments with the mechanism of light perception are, without doubt, highly speculative, and since for the most part they lack an experimental basis of any kind, they must remain of inter- est only as ingenious and interesting possibilities. In the case of the retinal pigment at least, it is possible to compare the responses to light and darkness with those exhibited by melano- phores in general (Parker ’06, p. 413)) and from our present knowledge we are unable to see either in the reactions of the retinal pigment or in those of the rods and cones anything more 182 LESLIE B. AREY than the presence of constant protoplasmic responses to a definite stimulating agent. a lack of uni- When the effect of temperature is considered, at once becomes apparent. formity of temperature upon the retinal Among the fishes the effect pigment is in agreement with Parker's ('06) general conclusion that in all melanophores, low temperature has the same effect as light and high temperature the same effect as In the retina of the living frog where this statement holds true only between the temperatures of 0" and 19°C. in the dark, notwith- standing the identical photic responses of the retinal pigment in fishes and amphibians, it is probable that a nervous control is responsible for the expanded condition both at low and at high temperatures; hence the responses in this animal are not comparable to those in fishes. Since only a limited temperature reaction occurs among fishes, and this in darkness as well as in lighi, it probably has no immediate adaptive significance, a tendency shown in but merely represents the survival of the responses of melanophores in genera1.l' In homothermous animals temperature, of course, can play no part in the normal activity of the retinal pigment, and even if the temperature responses in poikilothermous animals have an adaptive signifi- cance, it is evident that this particular advantage must be un- available to warm blooded vertebrates, in which keenness of sight is best developed. Moreover, the action of temperature upon the cone cells in the dark is variable. In the fishes, the cone myoids greatly elongate when warmed and shorten when cooled, while in the frog the cones are maximally shortened at high temperatures only and at all other temperatures remain unchanged. The inconsistent action of temperature in producing move- ments of the visual cells apparently has no common adaptive It should be pointed out, however, that in the case of the melanophores of the frog's skin, the reverse of this statement is true, since high temperature pro- duces similar effects to light, and low temperature to darkness. Below 38°C. temperature has practically no effect on the rate of deeom- position of the visual purple, as Kuhne ('79) showed, hence the changes of the pigment through the action of temperature are not related to this phenomenon. MOVEMENTS IN THE VISUAL CELLS 183 value, and, moreover, as in the case of retinal pigment, variable temperature can play no part in the normal movements of these cells in homothermous animals. It may be asked how it happens that high temperature in the dark produces diametrically opposed results upon the cone myoids of fishes and of the frog, when the responses to light are identical. The nervous system, presumably, is not involved in these reactions since temperature has a similar effect upon excised eyes. Between the minimal and optimal limits the movement of undifferentiated protoplasm is uniformly acceler- ated with increased temperature, and as has been shown in 135) the responses in length of the cones of another place (p. Abramis are markedly similar in this respect. Whatever the details of the process may be, it seems evident that among the fishes temperature acts purely as a physical agent in controlling the velocity of the reactions leading to positional changes of the cones. In frogs temperature apparently acts in an entirely different manner. The fact that only high temperature .is effective in producing a change, a shortening of the myoid, is best explained on the basis of Dittler’s theory of chemical stimu- lation, whereby increased temperature can be conceived of as favoring the formation of catabolic wastes which chemically stimulate the cones to shorten, while low temperature probably acts both in retarding catabolism, and by reducing the sensitivity of the myoid toward such products as are formed even under these conditions. A comparative study of the responses of the visual cells, throughout the various classes, to light and temperature re- veals difficulties in explaining the mechanism by which their positional changes are accomplished. Why do the rod cells of some animals shorten in the light whereas others lengthen? of birds lengthen through the Why do the rods of fishes and action of light, whereas the cones shorten? From the physiology of simple protoplasm, two alternatives are open. Either the myoids of the visual cells have become specialized to respond to certain stimulating agents in different ways, or, in various retinas the morphological structure of these 184 LESLIE B.. AREY contractile regions is such, that although the protoplasm re- sponds similarly in. all cases, the visible result upon the position of the entire cell is variable. The latter alternative would be realized if the active protoplasm were differentiated into ana- logues of myofibrillae which were arranged in some cases axially in the myoid and in other cases transversely or spirally. To become effective in elongating the myoid through contraction, a simple spiral ‘myofibril’ would have to make an angle greater than 45 degrees with the long axis of the myoid.l* In attempting to interpret the adaptiveness of the movements of the vertebrate retinal elements, it is evident from the fore- going discussion that neither with respect to the action of tem- perature nor of light has a satisfactory and constructive conclu- sion been reached. From the present state of our knowledge, therefore, the situation may be summarized in the following way. Although the movements of the visual cells and retinal pigment, when present, may have a certain unknown significance in connection with the mechanism of light perception, such movements can be interpreted at present only in terms of proto- plasmic responses to definite stimulating agents. 5. SUMMARY 1. The retinal pigment of the fishes studied requires 45 min- utes to 1 hour for light adaption and from 30 minutes to 1 hour for dark adaption. The cones of Abramis assume the light (shortened) position in 45 minutes, the dark (elongated) posi- tion in 30 minutes. Maximal elongation of the rods of Ameiurus in the light occurs in 45 minutes, maximal shortening in dark- ness in 30 minutes. 2. Both in light and in darkness, the retinal pigment of fishes shows greater expansion at a low than at a high temperature. High temperature is apparently more efficient in causing this 18 In connection with these possibilities mention will be made of only the fibrils found by Hesse (‘04) in both the inner and outer members of the rods and cones of several vertebrates, and of the longitudinal fibrils which Howard (’08) was able to trace throughout both the rods and cones of Necturus. Both workers, however, considered such structures as neiiroid in character. MOVEMENTS IN THE VISUAL CELLS 185 redistribution of pigment than is low temperature. The results obtained from the four fishes studied indicate that extreme to be conditions both of expansion and of contraction are not found in the retinal pigment of any one fish. 3. In darkness, the retinal pigment of the frog undergoes striking expansion between the temperatures of 0" to 14°C. and 19" to 33"C., whereas at the intermediate temperatures of 14" to 19°C. it is highly contracted. Temperature is without effect upon light-adapted retinas. Since the retinal pigment of the larvae of Rana catesbiana shows temperature responses identical with those characteristic of adults, there is no tem- porary larval recapitulation of the responses characteristic of fishes. 4. Light and darkness produce but limited changes in the distribution of the retinal pigment of Necturus. No definite effect of temperature could be detected. It is probable that the peculiar responses found in the frog have been developed within the anuran group. 5. The cone myoids of fishes shorten at low temperatures in the dark; at high temperatures they lengthen. Not only is elongation progressive, extreme conditions being found at 0" and 30" * C., but also the rate of change is directly proportional to the temperature gradient. Temperature is ineffectual in the light. 6. The myoid of the rods of fishes also elongates at high tem- peratures and shortens at low temperatures, but the extent of change is much less than that of cone cells. 7. The cone myoid of adult frogs in the dark shortens when subjected to a temperature of 19" to 33"C., but remains elongated at all lower temperatures. No definite temperature responses of the cone myoids were found in the larvae of Rana catesbiana. 8. A correlation between the length of the rod myoid and temperature was not detected in the frog either in darkness or in light. 9. The positions of the visual cells of Necturus are not affected by temperatures between the limits of 0" and 28°C. TEI JOURNAL OF C031PAR4TIVE NEUROLOCIT, 101,. 26, NO. 2 186 LESLIE B. AREY 10. In excised eyes of the four fishes studied, light causes a migration of the retinal pigment in Ameiurus only, whereas the pigment of none of the fishes moves in darkness. The rods of excised eyes of hmeiurus undergo movements both in the light and in the dark, the cones move in the light only. Neither exposure to darkness nor to light produces positional changes in the cone cells of excised eyes of Abramis orFundulus. Where tested, temperature was found to cause movements in the retinal pigment and cone cells in the excised eyes of fishes. 11. It is probable that the absence of responses in the excised eyes of fishes is due to an autoanaesthetiaation caused by the accumulation of catabolic products. 12. Dittler’s theory of the chemical stimulation of the cone myoid, propounded to explain the movements of the cone cells in isolated frog’s retinas, does not satisfactorily meet many conditions found in the responses of the cones of fishes. 13. The effects of temperature upon the rods, cones, and retinal pigment of the excised eyes of fishes are identical with those found in living animals, hence it is probable that temperature has a direct action upon these elements, its effect being physical in the sense that the chemical activity of the protoplasm is thereby accelerated to varying degrees. 14. Temperature has no effect upon the retinal pigment of the excised eye of the frog, therefore it is plausible that the action of temperature in living animals is physiological, whereby any adequate stimulus acting through the central nervous sys- tem can produce a striking pigment expansion according to the principle of specific energies. As in living animals the cone cell of the excised frog’s eye responds by a shortening at an elevated temperature only; it is probable that temperature acts directly upon the cone myoid, for this response, unlike that of the pig- ment, can not be interpreted by thc principle of specific energies. 15. Neither in the frog nor in Ameiurus are movements of the retinal elements evoked by exposure of the skin only to light. Hence the existence of an interrelation between dermal photosensitivity and the responses of the retinal elements by MOVEMENTS IN THE VISUAL CELLS 187 means of ‘retino-motor’ nerve fibers, as maintained by Engle- mann, is not substantiated. 16. Within the experimental limits at which fishes can be kept alive, the retinal pigment and visual cells are not affected by an increased or diminished oxygen supply. 17. Both in darkness and in light, and in excised as well as in normal eyes, carbon dioxide and ether completely check the movements of all the retinal elements of fishes. Chloretone and urethane, on the contrary, are inefficient in this respect. The action of carbon dioxide suggests that this may be the catabolic product that in many cases restrains the movements of the retinal elements when the circulation of the blood is interrupted. 18. Although the movements of the visual cells and retinal pigment, when present, may have a certain unknown significance in connection with the mechanism of light perception, such movements can be interpreted at present only in terms of proto- plasmic responses to definite stimulating agents. Cambridge, Muss., April 10, 1915. POSTSCRIPT A study of the influence of light on the movements of the frog’s rod has just been completed by the writer. Careful meas- urements prove that these elements are extended in the light and are retracted in darkness. Hence the results of the older workers (p. 123), who believed that the photic responses of the frog’s rod- myoid are the reverse of those occurring in fishes and birds, are not substantiated. 188 LESLIE B. AREY BIBLIOGRAPHY Papers marked ioith an asterisk have not beeti accessible in the original. .~NGELUCCI, A. 1878 Histologische Untersuchungen dber das retinale Pig- mentepithel der Wirhelthiere. Arch. f. Anat. u. Physiol., Physiol. Abth., Jahrg., 1878, pp. 353-386, 2 Taf. *I884 a Una nuova teoria sulla visione. Communic. prcvent,iva presentata all’ Accad. med. di Roma, 14 Juglio. 1884 b Una nuova teoria sulla visione. Gazetta Medica di Roma, 1884, pp. 205-210: 217-223. 1890 Untersuchungen iibcr die Sehthiitigkeit der Netzhaut und des Gehirns. Untersuch. zur Waturlehre d. Menschen u. d. Thiere (Mole- schott), Bd. 14, Heft 3, pp. 231-357, 2 Taf. *1905 Physiologie g6n6rale dc l’oeil. Encyclopedie frangaise d’oph- thalmologie, Tome 2, pp. 1-141. ARCOLEO, E. 1890 Osservazioni sperimcntali sugli elementi contratili della retina negli animali a sangue freddo. Bnnali d’Ottalmologia, Anno 19, Fasc. 3 e 4, pp. 253-262. The occurrence and the significance of photomechanical AREY, L. B. 1915 changes in the vertebrate retina-An historical survey. Jour. Conip. Neur., vol. 25, no. 6, pp. 535-554. BOLL, F. 1878 Zusatz zur Mitteil. vom 11. Januar, mitgeteilt in der Sitzung vom 19. Februar. Monatsber, d. konigl. preussischen Akad. d. Wis- sensch. zu Berlin aus dem Jahre 1877, pp. 72-74. CAE~LETOK, F. C. 1903 The color changes in the skin of the so-called Florida chameleon, Anolis carolinensis Cuv. Proc. Amer. Acad. Arts and Sci., vol. 39, no. 10, pp. 259-276, 1 pl. CHIARINI, P. 1904 a Changemcnts morphologiques, que l’on observe dans la r6tinc des vertebres par l’action dc la lurnicre et de l’ohscuritb. Prem- i&re Partie. La rbtine des poissons et des amphibies. Arch. ital. de Biol., Tom. 42, Fasc. 2, pp. 303-322, 4figs. *1904 b Cambiamenti morphologici che si verificano nella retina dei vertebrati per azione della luce e dell’ oscurita. Parte 1. La retina dei pesci e degli anfibi. Boll. della R. *4ccad. Mcd. di Roma, hnno 30, pp. 75-110. 1906 Changcnients morphologiqucs qui se produisent dam la. rdtine des vertehrbs par l’action de la lumi6r.c ct dc l’obscuritb. Dcuxi&me Partie. La rdtine des reptiles, des oiseaux et des mammifh-es. hrch. ital. de Eiol., Tom. 45, Fasc. 3, pp. 337-352, 8 figs. CZERNB, T’. 1867 nber Blendung der Netzhaut durch Sonnenlicht. Sit- zungsber. ti. Wiener Akad. d. Wisscnsch., Math-naturw. Kl., Bd. 56, -4bth. 2, pp. 409-428. 1 Taf. I)EUTSCHRIANN, R. 1882 Ober die Blendung der Xetzhaut durch directes Sonnenlicht. Arch. f. Ophthal., Bd. 28, hbth. 3, pp. 241-254. DITTLm, 11. 1907 Ober die Zapfenkontraktion an der isohertcn li’roschneh- haut. Arch. f. d. gcsam. Physiol., Bd. 117, IIrft 5 11. 6, pp. 295-328. 1 Taf. MOVEMENTS IN THE VISUAL CELLS 189 DTJBOIS, R. 1890 Sur la perception des radiations lumineuses par la peau, chez les ProtBes aveugles des grottes de la Carniole. Compt. rend. head. Sci. Paris, Tom. 110, pp. 358-361. EIGENMANN, C. H. 1900 The blind-fishes. Biological lectures; Marine Bio- logical Laboratory at Woods Holl, 1899, Ginn & Co., Boston, pp. 113- ENGLEMANN, T. \V. 1885 Uber Bewegungen der Zapfen und Pigmentzellen der Netzhaut unter dem Einfluss des Lichtes und des Nervensystems. (Nach 1884 gehaltenen Vortrag). Arch. f. d. gcsam. Physiol. Bd. 35, Heft 10, 11, u. 12, pp. 498-508, 1 Taf. EWALD, A,, UND KUHNE, W. 1877 Untersuchungen uber den Sehpurpur. Teil2. Entstehung der Retinafarbe. Untersuch. aus. d. Physiol. Inst. d. Univ. Heidelberg, Bd. 1, Heft 3, pp. 248-290. 1878 Untersuchungen uber den Sehpurpur. Teil 3. Veranderungen des Sehpurpurs und der Retina im Leben. Untersuchung. aus. d. Physiol. Inst. d. Univ. Heidelberg, Bd. 1, Heft 4. pp. 370-422. EYCLESHPMER, 9. C. 1908 The reaction to light of the decapitated young Necturus. Jour. Comp. Neur., vol. 18, no. 3, pp. 303-308. FICK, A. E. 1890 ifber die Ursachen der Pigmentwanderung in der Netzhaut. Vierteljahrsschrift d. naturf. Ges. zu Zurich, Jahrg. 35, pp. 83-86. FUJITA, H. 1911 Pigmentbewegung und Zapfenkontraction im Dunkelauge des Frosches bei Einwirkung verschiedener Reize. Arch. f. vergleich. Ophthal., Jahrg. 2, Heft 2, No. 6, pp. 164-179, 7 Taf. *GAGLIO, E. 1894 Le modificazioni del pigmento all’oscurith ed alla lucc nella retina della rana. Arch. di Ottalmologia, vol. 1, pp. 225-233. GARTEN, S. 1907 Die Veranderungen der Netahaut durch Licht. Graefe- Saemisch Handbuch der gesam. Augenheilkunde. Leipzig, Aufl. 2, Bd. 3, Kap. 12, Anhang, 30 pp., 5 Taf., 49 Textfig. GRABER, V. 1884 Grundlinien zur Erforschung des Helligkeits- und Farbensin- nes der Tiere. Prag u. Leipzig, 8 vo, viii + 322 pp. GRADENIGRO, G. 1885 Uber den Einfluss des Lichtes uncl der Warme auf die Retina des Frosches. Allg. Wicner med. Zeitung, Bd. 30, No. 29 11. 30, pp. 343-344, U. 353. GREEFF, It. 1900 Die mikroskopische Anatomie des Sehnerven und der Netz- haut. Graefe-Saemisch Handbuch der gesam. Augenheilkunde. Leip- aig, hufl. 2, Bd. 1, Kap. 5, 212 pp., 2 Taf., 53 Textfig. *HAMBURGER, D. J. 1889 Dorsnijding van der mrvus opticus bij Kikvorschen, in Verband met de Beweging van Pigment en Kegels in het Netvlies, onder den Invloed van Licht en Duister. Onderzoekingen d. Utrech- tsche Hoogeschol, Reeks 3, vol. 11, pp. 58-67. HERZOG, H. 1905 Experimentelle Untersuchung Bur Physiologie der Bewegungs- vorginge in der Netzhaut. Arch. f. Anat. 11. Physiol., Physiol. Abth., Jahrg. 1905, Heft 5 u. 6, pp. 413-464, 1 Taf. 1904 Uber den feinern Bau der Stabchen und Zapfen einiger Wir- HESSE, R. Zool. Jahrb., Suppl., Bd. 7, pp. 471-518, I Taf. beltierc. HOWARD, A. D. 1908 The visual cells in vertebrates, chiefly in Necturus Jour. Morph., vol. 19, no. 3, pp. 561-631, 5pls. maculosus. 190 LESLIE B. AREY KOR~NYI, A. v. 1892 Uber die Reizharlieit der Froschhaut gegen Licht und Wiirme. Ccntralbl. f. Physiol., Bd. 6, pp. 6-8. *KUHNE, 77;. 1877 Uher den Sehpurpiir. [Jntersuch. a. d. Phyiol. Inst. d. Univ. Heidelberg, Bd. 1, pp. 15-104, 1 Taf. 1879 Chemische Vorgiiiige in der Netzhaut. Ilermann’s Handbuch der Physiologie, Leipzig, Bd. 3, Teil 1, pp. 235-337. LEDERER, R. 1908 Veranderungen an den Stabchen der Froschnetzhaut unter Eiriwirkung von Licht und Dunkelheit. Centralbl. f. Physiol., Bd. 22, No. 24, pp. 762-764. *LODATO, G. 1895 Ricerche sulla fisiologia tlello strato neiiroepitheliale della retina. Arch. di Ottalmologia, vol. 3, pp. 141-148. LOEB, I., AND \~AsTENEYS, 1%. 1912 On the adaption of fish (E’undulus) to higher temperatures. Jour. Exp. ZoBl., vol. 12, no. 4, pp. 543-557. ~IAYER, P. 1881 Uber die in der zoologischen Station zu Neapel gehrauch- lichen Methoden ziir rnikroskoi,ischen Untersuchung. Mitth. Zool. Stat,. Xcapel. Bd. 2, pp. 1-27. MORANO, lg. 1872 Die Pigmentschicht dcr Pl’etzhaut. Arch. f. mikroslr. *Inat., Bd. 8, pp. 81-91, 1 Taf. MULLER, H. 1856 Anatomisch-pliysiologische Ilntersuchungen uber die Retina bei Menschen und Wirbelthiereri. Zeitschr. f. wisscnsch. Zool., Bd. 8,. Heft 1, pp. 1-122, 2 Taf. OVIO, G. 1895 Di un speciale axionc clclla cocain sulla funzione visiva. Annali di Ottalmologia, Anno 24, Suppl. a1 Fasc. 4, p. 23. PALMER, S. C. 1912 The numerical relations of the histological elements in the retina of nTect,urus maculosus (Raf.). Jour. Comp. Neur., vol. 22, no. 5, pp. 405-445, 12 fig. PARKER, G. H. 1905 The stimulation of the integumentary nerves of fishes hy light. Am. Jour. Physiol., vol. 14, no. 5, pp. 413-420. 1906 The influence of light, and heat on tho movement of the melan- ophorc pigment, especially in lizards. Jour. Exp. ZoBl., vol. 3, no. 3, pp. 401-414, 3 figs. *PICRGENS, E. 1896 Action de la IumiEre siir la rctine. Travaux tie Lab. de 1’ Inst. Solvay, Tome 2, pp. 1--38. 1899 Vorgange in der Netzhaut bei farhigcr Belichtung gleicher Intensitat. Zeitschr. f. Augenheilk., Bd. 2, pp. 125-141. POUCHET, G. 1876 Des changcmcnts de coloration sous l’influence des nerfs. Jour. de I’ Anat. et de la Physiol., pp. 1-90 et 113-165. SPAEm, It. -4. 1913 The physiology of thc chromatophores of fishes. Jour. Exp. Zool., vol. 15, no. 4, pp. 527-585, 4 pl. STORT, A. G. H. VAN GENDEREN 1886 Uber Form und Ortsveriinderungen dcr Elemente in dcr Sehzellenschioht nach Beleuchtung. Bericht iiber d. 18. Versamm. d. Ophthal. Gesell. zu Hcidelberg, 1886, pp. 43-49. 1587 Mouvemcnts dcs 616ments dc la rFtine sous l’influence dc i:l IumiBre. Arch. nderlandaises des Sciences exact et naturelles, publ. p. 1. Soc. holland. des Sciences, Tom. 21, Livr. 4, pp. 316-386. TREADWELL, F. P. AND HALL, W. T. 1905 Analytical chemistry, vol. 2. Wiley and Sons, New York, avo, xii+654 pp., 96 fig. EXPLANATION OF PLmm The figures of Plates 1 to 4 are photomicrographs; the figures of Plate 5 were drawn with the aid of a camera lucida. ABBREVIATIONS bac., rod my.con., cone myoid con., cone pd.cZ.pig., base of pigment cell con.acc., accessory cone prs.dst.bac., rod outer member ell.bac., rod ellipsoid prs.dst.con., cone outer member eZl.con., cone ellipsoid rtn., retina gtt.ol., oil globule scl., sclera mh.lim.ex., external limiting membrane st.nZ.ex., external nuclear layer my.bac., rod myoid st.pig., pigment layer 191 PLATE 1 EXPLANATION OF FIGURES The photographs in this plate, which show the influence of teinperature on the distribution of thc retinal pigment of fishes, are all at a magnification of 170 diameters. 1. Amciurus, 5°C. in the light. 2. Ameiurus, 25°C. in the light. 3. ilmeiurus, 5°C. in the dark. 4. Ameiurus, 25°C. in the dark. 5. Fundulus, t5"C. in the light. 6. Fundulus, 25°C. in the light. 7. Fundulus, 5°C. in the dark. 8. Fundulus, 25°C. in thc dark. 192 MOVEMENTS IN THE VISU.iL CELLS LESLIE 13. AREY 193 EXPLANATION OF FIGURES The photographs in this plate, which show t>he influence of temperature on the distribution of the retinal pigment of fishes, are all at a magnification of 170 diameters. 9 .$brnmis, 5°C. in the light. 10 Abramis, 25T. in the light. 11 'Abrnmis, 5°C. in the dark. 1% hbramis, 25'C. in the dark. 13 Carassius, 5°C. in t,hc light. 14 Carassius, 25°C. in the light. 15 Carassius, 5°C. in the dark. 16 Carassius, 25°C. in the dark. MOVEXEKTS IN THE VISUAL CELLS PLATE 2 I ESLII B. 4REY 195 EXPLANATION OF FICiUREil Figures 17 to 22, which are photographs showing the influence of teniperaturc~ on the distribution of the retinal pigment of the frog (adults and l:rrvac), are all at, a magnification of 170 diamcters. The larvnl Rana catesl)iana, from which figures 20 to 22 werr made, had a tot,al body length of 7.0 cni. A hits ,I2 hoino- gc:ncous immersion objective was uscd in inaking figures 23 xiid 24, which :we 715 diametrrs. xnagnifirtl 17 B. pipiens (adult), 3°C. in t,hc dark. 18 R.. pipiens (adult), 18°C. in the dark. 19 It. pipicns (adult), 33°C. in the dark. 20 R. cntesbianx (larva), 3°C. in the dark. 21 R. cateshi:tnn (larva), 16°C. in tlie dark. 22 R. catesbiann (larva), 32°C. in tlie (lark. 23 Carassius, 27T. in the dark. 24 Car:rssius. .yo(: in tlic (1;n-k. 196 MOVEVlEXTS IN THE VISUAL CELLS PLATE 3 LESIIE n. 4REY 197 PLATE 4 EXPL-%NATION OF FIGURES These photographs, t:tl\cii with a Lcitz ,'2 homogeneous imnicrsion objective, are at a magnification of 715 diamrters and shon tlw responses of the myoids of the cone cells of fishes nhen under the influence of tcmperature. 25 Ahramis, 3°C. in thc dark 26 Ahramis, 15°C. in the dark. 27 Abramis, 27°C. in the dark. 28 Fundulus, 3°C in the dark. 29 Fundulus, 27°C. in the dark. 198 hIOVEhlEn"1H lh' THE VISUAL CELLS I.ESL1E B. AHEY PLATE 5 EXPLANATION OF FIGUltES All drawings in this plate are at :L magnification of 930 diameters, a Leita 1?L homogeneous immersion objective tieing used. 30 Showing the positions of the rods and concs in a typical light-atlapt,ed rctina of Ameiiiriis. 31 Showing the positions of t)he rods and cones in a, typical dark-ndapted retina of Ameiurus. 32 Showing the effect of low temperature (3OC.) on the position of the dark- adapted rod- and cone-cells of Ameiurus. 33 Showing t,he effect, of high t,emper:tture (27°C.) on t,tic. position of the dark- adapted rod- and cone-cells of Ameiurus. 34 From the retina of a Rann pipiens that h:id I-)ccn previously kept at a temperature of 3°C. in the dark. The cone myoids remain elongated as at an intermediat,e temperature. 35 From the retina of a Rana pipiens that hat1 previously been kept, at) 18°C. in the dark. The cone myoids are elongated. 36 From the ret,ina of a Rann pipiens that had been kept at 33°C. in the (lark, showing the resulting shortening of the cone myoids. 200 prs.dst.con, 3Jl
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The Journal of Comparative Neurology
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Published: Apr 1, 1916
