THE PHOTORECEPTORS OF LAMPREYS

Transcription

1 229 THE PHOTORECEPTORS OF LAMPREYS I. LIGHT-SENSITIVE FIBRES IN THE LATERAL LINE NERVES BY J. Z. YOUNG, M.A. (Department of Zoology and Comparative Anatomy, Oxford.) (Received 25th January, 1935.) (With Three Text-figures.) CONTENTS. PAGE I. Introduction II. Reactions to illumination of whole animal III. Location of light-sensitive areas on the body IV. Connection of light-sensitive fibres with the lateral line nerves 234 V. Discussion VI. Summary References I. INTRODUCTION. IT was first shown by G. H. Parker (1905) that the tail of the ammocoete larva of lampreys is sensitive to light. He found the animals to be " negatively phototropic ", and by cutting out the paired eyes proved that these are not necessary for the reaction. Curiously enough he made no mention of the pineal eye as a possible photosensitive organ. Neither did he settle the question of whether the light produces a direct orientating effect on the animal, to produce what we should now call a topotaxis (Kiihn, 1919). The present research was therefore undertaken in order to discover: (a)whether the head as well as the tail is sensitive to light, and particularly, what may be the functions of the pineal complex; (b) the exact location of the light-sensitive areas on the tail, and their connections with the rest of the nervous system; (c) the nature of the taxis by which the animals avoid the light, that is to say the relation between the direction of the stimulus and response. The present paper deals mainly with the two latter problems, the responses which follow illumination of the pineal and paired eyes being described separately. The material consisted of larvae and adults of Lampetra planeri (Bloch), taken from the river Isis near Oxford, and adults of the larger species L. fluviatilis (L.), sent from Worcester. The larvae used were mostly of large size ( mm.), except for the newly-hatched individuals used for the experiments on p. 231.

2 230 J. Z. YOUNG II. REACTIONS TO ILLUMINATION OF WHOLE ANIMAL. In order to investigate the effects of illumination, a number of large ammocoetes were kept in a well-lit greenhouse, in a glass-sided tank with clean sides and bottom. When the tank was illuminated with weak, diffuse daylight the larvae all lay quiet, distributed evenly over the whole area of the bottom of the tank. In stronger daylight or electric light, however, they became restless and swam up and down. While swimming they keep close to the bottom, with the anterior end of the head rubbing against the angle made between the bottom and sides of the tank. This is a burrowing reaction and the larvae are in fact positively geotactic as well as negatively phototactic, the two reactions being rather difficult to separate. The tank was then arranged so that one half could be completely shaded and the other brightly illuminated by means of an electric lamp (Fig. 1), the greatest intensity of illumination being near the margin between light and shadow. After leaving the whole tank for some hours in total darkness, examination with a very Fig. 1. Diagram of tank arranged to show the reactions of ammocoete larvae to illumination, weak light showed that the ammocoetes were distributed irregularly over the bottom of the tank, and were lying at rest. The light was then turned on, and within less than s sec. all the animals in the illuminated half of the tank began to wriggle and soon started to swim. They moved up and down the tank, with their noses on the bottom in the usual way, and swam towards and away from the light with equal readiness. They crossed from light to darkness and vice versa, and often swam in and out of the illuminated area several times. Frequently, however, as they emerged from the darkness into the light, they were seen to accelerate and often to give a very strong burrowing reaction, so that the forward movement of swimming might be suspended for a few seconds, and the animal remain "burrowing" under the full intensity of the light. These more rapid and erratic movements on emerging from the light often resulted in the animal turning completely round and re-entering the dark area, but this could be seen to be due to random re-orientation during the burrowing movements and not to any orientating effect of the light. Several times the converse effect was obtained, namely of "burrowing" when entering the dark-

3 The Photoreceptors of Lampreys 231 ness, which also, in some cases, resulted in an "about turn" and re-entry into the illuminated area. After swimming about in this way for some minutes, the larvae began to settle down. When an individual came to rest in the dark area it remained there indefinitely, unless disturbed by the movements of its fellows; whereas no animal remained at rest for more than a few seconds in the illuminated area. In this way the larvae gradually became collected in the shaded part of the tank until all lay there, and so remained. The time required to reach equilibrium in this way was about 5-10 min.; it would undoubtedly be much less but for the fact that one swimming individual disturbs others; accordingly the time necessary for all to collect in the shade was shorter when fewer animals were present. The capacity to react in this way is present even in very young ammocoetes. Within 24 hours of hatching all the individuals in a bowl will start to wriggle on sudden increase of illumination. This behaviour is of a very simple type. It seems that the ammocoete may be regarded as having a single, undifferentiated photosensitive field, and as motor responses the movements of swimming and burrowing in whatever directions the surrounding mechanical conditions enforce. This conclusion, arrived at by study of the reactions of animals swimming freely in the water, has been abundantly confirmed by the detailed analysis of the light sensitive mechanism. III. LOCATION OF LIGHT-SENSITIVE AREAS ON BODY. In order to discover what part of the surface of the body is sensitive to light, the lampreys were placed, in the dark, in glass tubes of diameter slightly greater than that of their bodies, a current of water being passed through the tube. The animal then remained still, and could be observed for many hours consecutively. A small beam of light was provided by an electric bulb fixed in a large box camera, and was focused on to various parts of the body by means of either a lens or solid glass rod. The exposures were controlled by the shutter of the camera, and reaction times measured with a stop watch. The size of the beam used could be varied by means of the iris diaphragm of the camera. No attempt was made to investigate systematically the effects of light of varying intensities and in nearly all the experiments a 40-watt electric bulb was used; in a few cases, to try the effects of greater intensities, this was replaced by a Pointolite bulb. Using this technique, the most sensitive area was soon found to be the tip of the tail. When a narrow beam was shone on to the side of the tail at any level posterior to about the middle of the dorsal lobe of the caudal fin, then, after a latent period varying from \\ sec. upwards, the animal made violent wriggling movements. In order to investigate the latent period more thoroughly, the animals were illuminated at regular intervals, being left in the dark for either 1, 2, 5, 10 or 30 min. between each exposure. Considerable regularity over a number of exposures could be obtained. Thus in an experiment with L.fluviatilis, using 1 min. intervals and exposures of 1 sec, the animal reacted after the following latent periods: z\ sec, no reaction,

4 232 J. Z. YOUNG z\, 2}, 2\, 2\, 2\ sec, no reaction, 2{, 2%, 2\ sec. However, intervals of 1 min. are apparently too short for complete return to the resting state, and the reaction times gradually increased on successive exposures, as may be illustrated by the following series, from an ammocoete, using 1 min. intervals and illumination during the whole of the latent period: 2, if, 2, 2, 2, 2, 31, 2, 2\, 3$, \\, 3 sec. Hecht (1928) has shown that in the case of the light reactions of a variety of animals (Ciona, Pholas, Mya, etc.) the latent period is compound, being made up of an initial sensitization period of short duration, and a longer true latent period or maturation period, during which the products of the photochemical change are engaged in setting up impulses, and during which it is not necessary for the illumination to be continued. The photochemical mechanism in the tail of lampreys conforms to a similar scheme. With the intensities used, the minimum period during which illumination was found to be necessary was about - sec; if the light was switched off after such a period, then the animals moved after a total latent period of if 2\ sec. Exposures shorter than \ sec. failed to produce any movement, and longer exposures did not reduce the reaction time, which was never shorter than 1 f sec. This may be illustrated by the responses of an ammocoete: Illumination for 1 sec, moves after 2 sec. 1 sec, 25 sec. sec, 2jj sec.,, \ sec, no movement >»»» sec,,,,1,, 1 sec, moves after 2\ sec. \ sec, 2\ sec.,,,, \ sec, no movement opp t 1 sec, moves after 2% sec. There appears to be a relation between the area stimulated and the time and intensity of illumination necessary to produce a movement, though this interesting question has not yet been investigated in detail. Thus, shining on to the most sensitive part of the tail of a large ammocoete, a beam of 1 mm. diameter produced a reaction after 8 sec, one of 2 mm. diameter after 5 sec, and one of 4 mm. diameter after f sec. In order to discover what regions of the body and tail are sensitive to light, the beam was moved after each successive illumination, and it was found that the most sensitive area was limited to the upper part of the flank and the tail, from the level of the middle of the dorsal lobe of the caudal fin (Fig. 2). The fins themselves appear not to be sensitive, but they are so small that it is difficult to illuminate them alone. The ventral part of the tail is definitely less sensitive than the dorsal, and illumination from either above or below was followed by reactions only after longer exposures than were necessary using lateral illumination. Passing forwards from the tip of the tail, the times of illumination necessary to produce a movement became progressively longer, and in most cases no movement followed illumination of any part

5 The Photoreceptors of Lampreys 233 of the body anterior to the anus, except when the light was shone full on to the pineal region, in which case the ammocoetes almost always made movements, though only after latent periods much longer than those following illumination of the tail. The question of the location of the light sensitive tissue in the head is considered in a separate paper. For our present purpose, it is sufficient to note that the head is more sensitive to light than is the trunk, but much less sensitive than the tail. However, in a few cases illumination of the middle region of the body was followed by movements, especially when the light was shone directly on to the dorsal surface. For reasons which are given on p. 236, it is thought that these reactions are due to direct stimulation of the spinal cord by the light. Such reactions were not constant, and particularly could not be produced on a number of consecutive exposures, unless long intervals (more than 10 min.) were given. Thus the only area which is peculiarly sensitive to light is a line along the base of the caudal fin, and the whole question may be summarised in a specimen protocol in which most of the relative effects are illustrated. Fig. 2, Ammocoete larva of L. planeri, to show the effect of shining a light on to various parts of the body. See protocol on this page. Ammocoete, 125 mm. (MY). 17. vii Placed in tube with circulation of water and left in total darkness Shone narrow beam on to the following parts of the body, indicated on Fig. 2. Intervals of exactly 1 min. in darkness between each illumination. Point 1, movement after 2 sec. 2, i2f sec. 3, no movement in I min. 4, I min. S* *' " I mm - B, I min. Pineal eye, movement after 25 sec. Point 5, no movement in min. Pineal eye, movement after 26J sec. 54 sec. Point 4, no movement in \ min. 7..».» I min - l» I min. Pineal eye, movement after 54 sec. Point 8, movement after 25 sec. 5, no movement in \ min. Pineal eye, movement after 39 sec.

6 234 J- Z. YOUNG Results exactly parallel to those obtained with the ammocoetes were found when adults of L. planeri or the larger adults of L. fluviatilis were examined in the same way, the light sensitive area being found to be restricted to the head and tail. The adults have well developed paired eyes, and, as will be shown elsewhere, the animal moves after an illumination of these, though much less readily than after illumination of the tail. IV. CONNECTION OF LIGHT-SENSITIVE FIBRES WITH THE LATERAL LINE NERVES. (i) Section of the spinal cord. The analysis of the path by which impulses are conducted from the tail to the rest of the body began when it was noticed in an adult L. fluviatilis, in which the spinal cord had been cut, 1 that illumination of the tail was followed by movement of the head. This extraordinary phenomenon was confirmed in every case in which the spinal cord was cut, namely in thirteen ammocoetes of L. planeri and seven adult L. fluviatilis. Since in such animals only the head moves, it is very easy to see whether the first movement takes place towards the right or the left side. This is important in connection with the problem of whether or not the animal is orientated with reference to the direction of incidence of the light. The procedure was to illuminate the tail of the animal from one side, and to record the direction of the first swing of the head. As the accompanying figures show, there was no relation between the directions of illumination and of movement. This confirms the conclusions reached from study of the movements of free-swimming animals, namely that light activates, but does not orientate the lampreys. Thus an ammocoete of 145 mm., in which the spinal cord had been cut on April 2nd, 1932, was examined on April 6th, 1932, the light being shone at oneminute intervals as follows: On right side of tail, head moves after 2 sec. towards left.,. left )i,. left )»., right»» right.. left,, ICIL M,,.. left,, leit,,,,, ( 2% sec. z\ sec. 3 sec. 25 sec. z\ sec. 2\ sec. 5 sec. 35 sec 4 sec. 3 sec.,, left. left. left. left. Therefore five times the first movement was towards the side stimulated, six times it was in the opposite direction. 1 The operation is very easy to perform on account of the absence of vertebrae, and the nnimals survive it indefinitely. The result is a remarkably profound spinal shock. Eventually the body becomes capable of producing slow metachrone waves when strongly stimulated, but nothing like effective swimming is ever regained.

7 The Photoreceptors o* Lampreys 235 Since the impulses set up by the photochemical processes in the tail are not conducted through the spinal cord, there remain only two possible paths by which the head might be affected: (1) by some hormone secreted into the blood, (2) via the lateral line nerves. The first possibility is almost excluded by the time relations, whereas the following experiments show the second alternative to be correct. (ii) Section of lateral line nerves. This operation was first successfully accomplished on adult L. fluviatilis under urethane anaesthesia. A longitudinal middorsal incision was made just behind the gills, and continued downwards until the endorhachis ("dura mater") surrounding the spinal cord was reached. By the use of a low-power microscope and dissection of the muscles from the outside of this membrane the lateral line nerve can be discovered and cut on each side, the wound being then sewn up in the usual manner. The result of this operation, which was performed on four adult L. fluviatilis, was entirely to abolish the sensitivity of the tail to light. Later the technique was perfected to allow of the severance of the lateral line nerves of ammocoetes and adults of L. planeri, the operation being performed on seventeen larvae and two adults. The result was again that all sensitivity of the tail was abolished, except in two cases, in both of which histological examination later showed that one of the lateral line nerves had not been completely cut. The animals were examined at intervals after the operation for signs of regeneration, but the sensitivity of the tail never reappeared, although the ammocoetes were kept for periods up to 184 days, and the adults up to 63 days. Histological examination of the stumps of the nerves also showed that regeneration proceeds very slowly. 1 In four experiments with L. fluviatilis, and four with ammocoetes of L. planeri, the lateral line nerve was cut on one side only, and after this operation, it was found that the animal still moved when the tail was illuminated from either side, though sometimes, not always, longer exposures were necessary for illumination of the operated than for the normal side. This strongly confirms the conclusion reached above, that the tail constitutes an undifferentiated light-receptive field, and that illumination from one side or the other does not produce orientation of the animal. This was further confirmed by placing such animals, operated on one side only, in round bowls in the dark, and then illuminating them. If there were any orientating effect of the light, they would be expected under such conditions to show forced movements, that is to say, to swim continually in one direction or the other. In fact, they swam equally frequently in right- and left-handed directions. When such animals were examined by illumination in a tube, it could be seen that the first movement of the head might take place either to the right or to the left. All of these experiments confirm the conclusion that the reaction to illumination is a photokinesis, and not a topotaxis. A curious feature of most experiments in which the lateral line nerves were cut was that the region in which the operation had been performed became sensitive to light. This sensitivity was particularly remarkable in that the latent period of the reaction approximated in shortness to that seen following illumination of the tail. 1 All the experiments were performed during the winter montht.

8 J. Z. YOUNG However, a difference lay in the length of the adaptation period of darkness which was necessary between illuminations, this being much longer than the one minute or less needed for the responses from the tail. At first it was thought that this sensitivity was due to regenerating fibres of the lateral line nerve, but this explanation was abandoned when it was noticed that the reaction could be elicited on the day after the operation. The impulses are in fact set up by the effect of the light on the spinal cord itself, which has been made accessible to the light by removal during the operation of the layers of pigment beneath the epidermis and around the spinal cord. This was proved by two experiments in which the spinal cord was scraped free of pigment dorsally, without section of the lateral line nerves, the operated region becoming immediately sensitive to light. Fig. 3. Section of lateral line organ from tail of adult L. plaiteri, to show pigment (p) in the epidermal cells. Outlines filled in over photograph taken with Zeiss apo. imm Bouin, Ehrlich's haematoxylin. This also provides a possible explanation for those rare cases in which illumination of the back of an animal was followed by movements (p. 233). Presumably in such animals the pigment was not sufficiently dense to prevent light affecting the cord. Thus incidentally, this case provides a very clear demonstration of the need of internal pigmentation for the protection of the tissues of animals from light. It is clear from the experiments described that the impulses initiated in the tail by changes of illumination are carried forward in the lateral line nerve'. It remains to demonstrate the site of the initial photochemical processes. The lateral line organs extend down the side of the tail, close to its dorsal margin, and this is exactly the region which is most sensitive to light. Examination of sections of these caudal neuromasts showed that the epidermal cells surrounding them often contain granules of yellow pigment (Fig. 3), which may perhaps be concerned with the photosensitivity of the tail. However against this possibility must be set the facts that (a) 1 Parker (1905) claimed that movements could be obtained on illumination of the tail e\en after decapitation. I have tried this experiment repeatedly, but have never been able to confirm his observation.

9 The Photoreceptors of Lampreys 237 the pigment was not found in all the tail neuromasts examined, and (b) that it occurs, though rarely, in the neuromasts of the head. Further work is therefore required to demonstrate conclusively whether it is connected with the reactions to light. V. DISCUSSION. Detailed investigation has thus fully justified the first impression that the behaviour of lampreys, and particularly of ammocoete larvae is extremely simple. They seem to have only one type of movement available, namely, swimming forwards and downwards, the burrowing movements being simply modified swimming. The effect of light on the ammocoetes is mainly on the tail, which is extremely sensitive, the photoreceptors being connected with the rest of the nervous system by the lateral line nerve. This mechanism plays a large part in the normal behaviour of the lamprey, causing it to swim when it is illuminated. It also materially assists in causing the animal to bury itself, as is easily seen by placing specimens in a bowl with mud in the total darkness. The downward burrowing movements will cause them to bury the head and most of the body, but many tails will be left protruding. However, if a light is then turned on, the tails also are withdrawn below the surface. After metamorphosis, the same photoreceptor mechanism in the tail remains in action, causing the animals to swim around when they are illuminated; since, however, they no longer swim head downwards, they do not bury into the mud. Moreover, the presence of the paired eyes in the adults introduces new possibilities of telotaxes, and perhaps reactions to formed stimuli, although these interesting possibilities still await investigation. It has been shown by a variety of observations, that the effect of light on the larval lampreys is simply to stimulate them to swimming movements, and that it does not serve to orientate them with reference to the direction of the light. We may express this by saying that the reaction is a photokinesis (Eigenmann) and not any form of topotaxis (Kiihn, 1919). Such photokinesis constitutes an extremely simple type of behaviour, in that the light itself has no orientating effect on the animal, but simply sets off a motor mechanism. We may thus compare it with the avoiding reaction (phobotaxis) of Jennings, and with such reactions as the sudden withdrawal of worms into the mud when illuminated. It is interesting to find such a simple type of behaviour among Chordates, since in most of these the reactions through the eyes involve topotaxes and usually also reactions to formed stimuli. However the skin has been shown to be sensitive to light in certain blind fishes, Chologaster and Amblyopsis (Eigenmann, 1900), though not in normal Selachians or Teleosts (Parker, 1909; Scharrer, 1928). The experiments of Eigenmann seem to indicate that the mechanism by which his blind fish collect in the dark is similar to that of ammocoetes. On the other hand, Parker (1903) showed that blinded frogs respond to illumination by turning towards the light, so that, in that case, the skin photosensitivity provides a topotactic mechanism.

10 238 J. Z. YOUNG VI. SUMMARY. 1. The tail of larval and adult L. planeri and adult L. fluviatilis contains a photoreceptive mechanism involving an initial short sensitization period, and a latent period. 2. The impulses initiated by the photochemical change are carried in the lateral line nerves, as is shown by the facts that section of these nerves abolishes the response, whereas section of the spinal cord does not do so. 3. Motor responses are only occasionally seen after illumination of parts of the body other than the tail. These responses are apparently due to direct stimulation of the spinal cord, and can be regularly elicited if the pigment protecting the latter be removed. 4. Motor responses may follow illumination of the head, either of larvae or adults, but only after illumination periods much longer than are necessary to obtain a response from the tail. 5. The responses play a part in the normal behaviour of the animals by assisting them to bury themselves completely in the mud. 6. The stimulus of illumination of the tail simply initiates swimming movements, and there is no orientation of the animal with reference to the direction of the light. This is confirmed by the observation that, following illumination of the tail from one side, the first movement of the head may be either towards or away from the side stimulated. Further, after section of one lateral line nerve only no forced movements occur on illumination. The reaction may thus be described as a photokinesis, and does not involve any true topotaxis, its effect is to prevent the animal remaining in any illuminated area. My thanks are due to Prof. E. S. Goodrich, F.R.S., for reading the manuscript of this and the following paper and for his encouragement and advice during the course of the work, also to Mr W. E. Young for assistance in some of the experiments. REFERENCES. EIGENMANN, C. H. (1900). "The blind fishes." Biul. Led. Marine Biol. Lob. Wood's Hole, p HECIIT, S. (1928). J. gen. Physiol. 8, 291. KOHN, A. (1919). Oie Orientterung der Tiere im Raum. Leipzig. PARKER, G. H. (1903). Amtr.J. Physiol. 10, 28. (1905)- Amer.J. Physiol. 14, 413. (1909). Amer. J. Physiol. 25, 77. SCHARRER, E. (1928). Z. vergl. Physiol. 7, 1.