The Central Nervous Control of Colour Change in the Minnow (Phoxinus Phoxinus L.): I. Blinding and the Effects of Tectal Removal On Normal and Blind Fish^*

Except for a few observations by von Frisch (1911), Dijkgraaf (1949), and Wichers (cited by Healey, 1957), no work has been done on the central nervous control of colour change, anterior to the medulla oblongata. Von Frisch performed a series of experiments in which he stimulated various parts of the minnow brain. After negative results from the cerebellum and the optic tectum he concluded that there might possibly be a darkening centre in the diencephalon. Further evidence for a possible diencephalic centre was provided by Wichers who found that the minnow could change colour after removal of both the optic tectum and the eye on the same side. Because of the complete chiasma this would suggest that the paths from the retina avoid the optic tectum. Dijkgraaf, however, found that complete tectal removal from a fish which had darkened as a result of bilateral optic nerve section resulted in pronounced paling. From this work of Dijkgraaf it would appear that the optic tectum is involved in chromatic adaptation.

The experiments to be described here are concerned with the colour of minnows following optic nerve section and tectal ablation and with the role of the optic tectum and the fibre pathways involved in colour change.

The minnows used were adults, 6 cm in length, of both sexes collected from the River Lea in Hertfordshire. They were kept throughout the year in large sinks where the temperature ranged from 15 to 20°C.

The colour of the fish was recorded using the Derived Ostwald Index (D.O.I.) developed by Healey (1967). The general tint of the dorsal surface of the fish was compared with a series of nine standard grey tints, very light grey being o and very dark grey 8. In order to test the extent of their adaptation to a black or white background the fish were placed in 1 1 glass beakers containing water to a depth of 8 cm, and these beakers were placed in larger metal containers painted black or white and also containing water. The illumination was by means of a 40 W frosted tungsten-filament bulb suspended 45 cm above the surface of the water. The background was changed by transferring the beakers from one metal container to the other with the minimum disturbance to the fish. During the experiments in which the fish were kept for long periods on black or white backgrounds they were placed in glass experimental aquaria. These aquaria measured 87·5 × 50 × 55 cm, were painted black or white on the outside, and partitioned internally into four equal compartments by means of black or white Perspex walls provided with holes to allow adequate circulation of water.

The surgery was performed under a 0·008% solution of MS. 222 (Tricaine methane-sulphonate, Sandoz, Ltd.) using fine knives (J. Weiss and Co. London). Following tectal ablation the hole produced by the partial removal of the frontal and parietal bones was closed with Simplex acrylic denture repair material (Dental Fillings Ltd., London).

Lesions were made in the optic tectum using a technique of micro-chemical cautery similar to that used by Clark & Scott (1962) on the diencephalon of the frog. The lesions were placed by hand using fine glass micro-pipettes filled with dichloracetic acid. Their position was determined by an eye-piece graticule in the eye-piece of a dissecting microscope. The graticule was a simple calibrated grid which enabled the position of the lesion to be marked on a drawing of the brain on squared paper.

At the completion of each experiment involving brain lesion or ablation the brain was removed and fixed in formol-saline, embedded in paraffin wax, sectioned and stained using cresyl violet or the method of Klüver & Barrera (1953).

Blinding was carried out on each side by making a small incision in the skin near to the eye so that the latter could be rotated until the optic nerve could be clearly seen and cut. On recovery from the anaesthetic the fish paled to a D.O.I. value of 1·5 but soon began to darken to a value of 2−3 after 30 min. This darkening continued throughout the next 24 h to reach a value or 6·5−7·5. The results from 14 fish following blinding were recorded for 59 days (Table 1).

It would appear that the population is composed of two types, those which are dark 24 h after blinding and remain so, and those which are dark 24 h after blinding but subsequently lighten after a variable period. The fish which paled maintained their colour relatively constant for long periods, but did not go paler than D.O.I. 2 or 3.

No gross histological differences in the central nervous system could be found between the pale and the dark fish after being blind for 6 weeks. Experiments were performed in which the fish were subjected to various light intensities and different backgrounds but all failed to produce any change in the tint of the skin.

The optic tectum was completely removed from 15 fish, the skull was sealed, and the fish was placed in an experimental aquarium. Four fish were killed after 3 h, and the colour of the remainder was recorded at intervals on both black and white backgrounds for a period of 11 d, after which these fish were also killed. Of the 11 fish only three gave useful results, the rest showing various degrees of degeneration in the brain. This degeneration produced large amounts of necrotic tissue in the mesen-cephalon and diencephalon in which no definite structures were clearly visible.

The results therefore relate only to four fish killed after 3 h and three fish killed after 11 d. In these seven fish the tectum was completely removed and, as far as could be seen, the rest of the brain, including the geniculate complex, was intact. All the fish paled within 24 b after operation, the degree of paling varying from 1 to 4·5 D.O.I., with a mean of 2·7. This tint was maintained for almost 24 h without change. These results confirm the observations of Dijkgraaf (1949).

The chromatic behaviour of the fish which survived for 11 d is shown in Fig. 1. None of the three showed any maintenance of the initial pale colour but darkened regardless of background. The darkening began 24 h after operation and continued steadily up to the seventh day after which it levelled off. The final colours of the fish differed from one another but were not near the possible maximum extremes of the chromatic range.

The optic tectum was completely removed on the right side in five fish. These were allowed to recover and then tested after 1 h. In every case the fish could perform normal colour change in both extent and direction.

In ten fish the left optic nerve was cut and the animals were allowed to recover. On the following day five of the fish had the tectum removed from the left side and five from the right. All ten fish showed normal colour change in both extent and direction (Fig. 2). Control fish blinded unilaterally by optic nerve section were tested with only the skull bones removed and the skull cemented over, and in all five cases they could perform normal colour change.

Eight minnows were blinded by cutting the optic nerves on each side and were allowed to recover and darken for 24−48 h, by which time they had reached D.O.I. values of 6·5−7·5Five of these darkened blinded fish then had the optic tectum completely removed and three control fish had the skull bones removed without damage to the tectum. In all the fish with the optic tectum removed, but in none of the controis, marked paling occurred to give D.O.I. values of 1−1·5 within 2 h.

In a group of 34 fish, following bilateral section of the optic nerve 24−48 h previously, the left lobe of the tectum was removed. On recovery a few of the animals showed a slight paling but the majority did not show any difference from their pre operative condition.

Four fish 24−48 h after bilateral section of the optic nerve had more local tectal ablations, the extent of the areas removed being shown in Fig. 3.

• This fish had the anterior part of the tectum bilaterally removed. The fish stayed fully dark throughout the whole of the observation period of 3, h and maintained a constant D.O.I. of 7.

• The posterior part of the tectum was bilaterally removed and 2 h after the operation had reached a D.O.I. of 1·5. Although the dorsal tectum had been removed there still remained a part of the posterior lateroventral tectum with intact connexions to the torus asemicircularis.

• The dorsal part of the tectum in the posterior region was removed leaving the lateral part intact. The fish had an initial D.O.I. of 7 and a final D.O.I. of 2·5.

• The dorsolateral part of the tectum in the posterior region was removed. The fish had an initial D.O.I. of 7 and a final D.O.I. of 2·5.

The findings from this experiment show that the removal of the dorsal part of the posterior tectum results in complete paling of the blind fish.

Normal blinded fish were anaesthetized, the left lobe of the optic tectum was removed and the fish were allowed to recover. After the fish had been re-anaesthetized the right posterior part of the tectum was exposed and the lesions were made. The lesion was cylindrical in shape and passed completely through the optic tectum. The diameter of the lesion was 0·2 mm ( ± 0·04 mm). In making the lesion the apex of the cerebellum was taken as the reference point because of its relatively constant position with respect to the rest of the tectum. The anterior−posterior axis is called the X-axis and the lateral axis the T-axis, the cerebellar apex forming the zero point of both axes (Fig. 4). Lesions anterior to the cerebellar apex are given positive values while those that are posterior have negative values.

The final D.O.I. was recorded at least 2 h after recovery from the anaesthetic. The final value subtracted from the original D.O.I. is referred to as the degree of paling (d.o.p.). The lesion positions and the d.o.p. are summarized in Fig. 4.

The steps of the D.O.I. scale are not equivalent and the d.o.p. values given cannot be taken as having a quantitative significance. They merely indicate very roughly the degree of paling that followed the lesion. In normal fish colour change following background reversal from black to white results in a d.o.p. of 6 which is only 2 degrees higher than the d.o.p. of 4 produced by lesion X0 Y1 mm. There appears to be a small region in the dorsal posterior part of the tectum whose removal results in almost maximal paling in blind fish. The lesions Xo Kimm and Xo Y1·33 mm represent the centre of the region giving values of d.o.p. of 4 and 3 respectively. These two lesions are bordered by a group of lesions giving values of only 1, these being X0 Y0·66mm, X+0·33 Y1·33mm, X−0·25 Y1·33mm, +0·1 Y1·66 mm

Control lesions were placed using the micro-pipette filled with Ringer to test the effect of the acid. These yielded the same results as with the acid but the extent of the lesion was more difficult to determine. The question as to whether the acid destroys more than is immediately visible is one which applies to all methods of lesion-making. In the present experiment the brain was constantly washed with Ringer during the placing of the lesion to prevent the spread of the acid. In fish which had been left for 16 d following lesion the lesion was observed to be fully healed.

Minnows were found to change colour normally when blind on one side regardless of whether the blinding was accomplished by the removal of the optic tectum or by cutting the optic nerve. Following the total removal of the optic tectum, the fish went pale, as reported by Dijkgraaf (1949) but unlike the fish used by the Dijkgraaf they could not perform colour change in response to background tint, from which it must be con-cluded that the tectum is essential for chromatic adaptation. The observation by Wichers (reported by Healey, 1957) that the minnow with one eye removed and the optic tectum removed from the same side, could change colour was confirmed. Further observation of the preparation showed the colour change to be normal in both rate and extent. The removal of one eye and one lobe of the tectum on the same side means that the fish is without direct retino-tectal fibres, whereas the optic tectum is necessary for chromatic adaptation. What is needed is for some intermediate body to be present which could relay the information gathered from the retina to the tectum. From the anatomical study of the optic system of the minnow (Gentle, 1968) the only body which could perform this function is the geniculate complex. The geniculate complex of the minnow receives numerous fibres from all parts of the retina and sends numerous fibres to the optic tectum and to the geniculate complex and the tectum of the opposite side.

Von Frisch (1911) found that section of the brain above the level of the medulla always resulted in paling and concluded there was a paling centre present in the medulla which worked through the autonomic nervous system. The removal of the tectum has the same final effect as cutting the brain above the medulla in causing paling. One may therefore suppose that the tectum can act on the medulla by inhibiting it, with the resulting darkening. The paling of a blind or normal fish following tectal removal is not at the same rate as the paling to a white background and requires at least 2 h. Furthermore, this pallor following tectal removal is not maximal and is not maintained for periods of longer than a few hours. From these considerations it would appear that the tectum may not only inhibit the paling centre but may also excite it, and that the chromatic adaptation of the fish may include an excitatory-inhibitory action of the tectum on the medullary centre.

The removal of the tectum in the blind minnow may lead to paling because it frees the medullary centre from the control of the tectum. This would in turn, imply that in the blinded state the tectum completely inhibits the centre. The inhibition is not removed when one lobe of the tectum is removed, which means that the tectum on one side can control the medullary centre on both sides. The inhibition cannot be removed by removing the anterior region of the tectum in both lobes; rather it appears to be localized to a small region in the dorsal posterior part of the tectum. The question arises as to whether this represents a definite medullary controlling region, or whether the removals and lesions have destroyed the fibre connexions as they pass out of the tectum. In view of the lack of any conclusive evidence of localization of function in the optic tectum and the fact that the lesion was in a region where a large number of efferent fibres leave it, it seems most likely that the lesions produced their effect by destroying the fibres as they pass out.

The author wishes to express his thanks to Professor N. Millott of the Department of Zoology, Bedford College, London who kindly provided laboratory facilities and to Dr E. G. Healey who suggested the topic, supervised the work, and offered much encouragement and advice. Finally, he is indebted to the S.R.C. for financial support during the tenure of their Studentship.