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Monocular imprinting and regional incorporation of tritiated uracil into the brains of intact and ‘split-brain’ chicks
Brain Research, 56 (1973) 227-237
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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
MONOCULAR IMPRINTING AND REGIONAL INCORPORATION OF TRITIATED URACIL INTO THE BRAINS OF INTACT AND 'SPLITBRAIN' CHICKS
G. H O R N , S. P. R. ROSE AND P. P. G. BATESON
University of Cambridge, Department of Anatomy, Cambridge, Department of Biology, Open University, Bletchley, Bucks. and University of Cambridge, Sub-Department of Animal Behaviour, Madingley, Cambridge (Great Britain) (Accepted December 6th, 1972)
SUMMARY
When intact chicks with a patch over one eye were exposed at a post-hatch age of 14-19 h to a flashing light, no regional differences between the two hemispheres were found in the incorporation of tritiated uracil into acid-insoluble residues. Similar biochemical studies were made using 12 chicks in which the supraoptic commissure had been transected. Each chick had one eye covered with a patch and was exposed to a flashing yellow light for a total of 60 min. It was subsequently given two choice tests between the familiar flashing yellow light and an unfamiliar flashing red light, first with its trained eye alone exposed and then with its untrained eye alone exposed. All of the 12 chicks approached the familiar flashing yellow light with the originally trained eye uncovered, but not with the originally untrained eye uncovered. The rate of incorporation of tritiated uracil into presumed RNA was 15.2 ~ higher in the trained side of the forebrain roof than in the untrained side. No other regional differences between trained and untrained sides were observed. The results show that certain non-specific consequences of the imprinting procedure, such as general changes in hormonal levels as a result of stress, non-visual sensory stimulation and motor activity, cannot account for the observed biochemical changes in the forebrain roof.
INTRODUCTION
When newly hatched domestic chicks are exposed to a conspicuous object they rapidly learn its characteristics and subsequently may approach it in preference to
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dissimilar objects. The learning process by which young birds form a social attachment for an object to which they have been exposed is called 'imprinting'. We have tound that when domestic chicks are exposed to a highly-effective imprinting stimulus, a rotating flashing light, changes occur in the presumed rates of protein and RNA synthesis in the brain4.5, 26. Of special interest was the discovery that after 76 min of exposure a sharp increase in the incorporation of tritiated uracil into acid-insoluble substances occurred in the upper part of the forebrain alone. Such effects were not found in chicks exposed to a flashing light for a shorter period of time, in a control group kept in the dark, or in chicks exposed to a steady level of background illumination for the same length of time. A number of factors could account for the biochemical differences between the experimental group exposed for 76 min and the other chicks. The groups may, for example, have differed in the amount of motor activity and in the levels of stress to which they were variously subjected. Protein and RNA metabolism in the central nervous system may be affected by motor activity xa,ls, by stress 1,27, and by exogenous A C T H 17A9 or corticosteroneL The experiments using exogenous hormones suggest that any procedure which modifies the endogenous levels of these substances may have repercussions on RNA and protein synthesis in the brain. Such general effects, which may often accompany learning, can largely be allowed for by effectively restricting input to one side of the brain during the training procedures. If any biochemical differences exist between the 'trained' and 'untrained' sides of the brain, it is reasonable to ascribe them to differences in visual experience. They are unlikely to result solely from general changes in hormonal levels as a consequence of stress, to differences in non-visual sensory stimulation, or to differences in motor activity between the corresponding sides of the body. Interhemispheric transfer takes place when only one eye is exposed during the imprinting of intact Peking ducklings 24. Similarly, a pilot study indicated that after exposure to a flashing light, day-old domestic chicks with one eye occluded showed a preference for that light when the untrained eye was subsequently tested (Bateson unpublished). Moreover, transfer of an acquired aversion to pecking at objects treated with methyl anthranilate has been observed in young chicks 9. All these studies suggest that both sides of the brain of the intact chick are trained when input is restricted to one side. Therefore, in the first series of experiments described here, we studied intact chicks which had one eye occluded. No differences between stimulated and unstimulated sides were found and so, in the second series, attempts were made to interrupt transfer from one side of the brain to the other. Transfer of a visual discrimination task is severely impaired in pigeons, the optic chiasma of which is completely crossed 1°, following transection of the supraoptic commissureZl,2L It seemed reasonable, therefore, to suppose that this surgical procedure would impair interhemispheric transfer in chicks which had one eye occluded when exposed to an imprinting stimulus. In the second series of experiments described here the incorporation of tritiated uracil into presumed RNA was studied in 'split-brain' chicks which had had only one eye exposed during the imprinting procedure. A preliminary account of the second series of experiments has already been publishedtL
IMPRINTING IN 'SPLIT-BRAIN' CHICKS
229
MATERIALS AND METHODS
Intact chicks The first series of experiments were conducted on unoperated Chunky chicks which were hatched and reared in the dark. Each chick had an opaque rubber cap (12 mm in diameter × 3 mm deep) fitted over one eye. The cap was attached round the eye with a rubber-based false eye-lash adhesive ('Eyelure'). Subsequently the chicks were marked, weighed, and, between 14-19 h after hatching, given an intracardiac injection of 20 #Ci [5-3H]uracil (specific activity 1000 mCi/mmole, Radiochemical Centre, Amersham, Bucks.) in 0.1 ml of Locke solution. Handling of the chicks was kept to a minimum and was always done in the dark or with the aid of a dim green light. Each chick was killed by decapitation 150 min after it had been injected. In the first experiment 12 chicks from the same hatch were used. Half had a cap fitted over their left eye and half over the right eye. After injection each chick was placed for 37 min in a dark incubator kept at 29-30 °C. It was then transferred to a training pen and exposed to an orange flashing light for 76 min. Details of the apparatus and methods are given elsewhere 5. After exposure the chicks were returned to the dark incubator for 35 min, given a 2 rain test and then killed. In the second experiment of this series, 11 chicks from one hatch were treated in a similar way but were exposed for 155 min to the flashing light. The period of exposure started 5 min before injection. In this experiment 6 chicks had the right eye exposed and 5 the left eye; originally the number was 6 but one chick lost its patch during the experiment and was discarded. As in our previous studies, the brain was dissected into forebrain roof, forebrain base and midbrain. The 3 regions were then divided down the median plane. The regions were placed into coded specimen pots and rapidly frozen on solid CO2. They were kept frozen until biochemical analyses were performed on each sample. The codes were not known by those performing the analyses. The samples were treated as before 5. Specific activities, expressed as disint./min/mg protein in the sample of the acid-insoluble fraction varied considerably between chicks. Values for all 6 samples from a brain were therefore standardised as percentages of the mean specific activity for that brain. As protein values obtained for any region did not vary significantly with behavioural treatment, expressing the results in this way was taken as a measure of incorporation into presumed RNA. Total radioactivity (pool) consisting of radioactivity in the acid-soluble plus the acid-insoluble fractions was measured and standardised in the same way. To assess the validity of the measures of specific activity used, the time-course of uptake and incorporation of [all]uracil into the brains of day-old chicks was studied in an earlier experiment. Each chick received an intracardiac injection of 20 #Ci [3H]uracil and they were killed at varying times afterwards. Uptake, as measured by changes in total pool, increased linearly for the first 60 min and then levelled off, remaining constant for at least a further 90 min. Incorporation into acid-insoluble substances proceeded linearly for at least 150 min, the activity of acid-insoluble relative to total pool at the end of this time being less than 6 ~ .
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"Split-brain' chicks In the second series of experiments Chunky chicks were hatched and kept in a dark incubator for 1--4 h after hatching. Each chick was weighed and anaesthetized by an intraperitoneal injection of 0.1 ml of 'Equithesin'. The cartilaginous skull was incised in a parasagittal plane approximately 3 mm to the left of the midline. A flap of cartilage extending to approximately 1 mm beyond the midline on the right was separated from the underlying dura and folded down. The dura overlying the left hemisphere was incised and reflected to expose the interhemispheric fissure. Following a technique similar to that described by Cu6nod and Zeier 11 a fine knife was lowered into the fissure, damage to the sagittal sinus being avoided by applying gentle traction on the dural flap. The blade was lowered to the best setting for the supraoptic commissure. This setting was determined by direct observation of the position of this commissure in a preliminary series of experiments and was found to be 2.0 mm anterior and 2.5 mm above the interaural plane in chicks weighing 35-50 g. These coordinates refer to a chick which is held in the stereotaxic apparatus with the upper surface of the beak bar, inserted between upper and lower beaks, set 2.7 mm below the interaural plane. After positioning, the knife was moved approximately 3 mm in the sagittal plane and slowly withdrawn. The dural and cartilaginous flaps were replaced and the skin wound closed. The surgical procedures were conducted using an operating microscope. Atter operation, the chick was maintained in a dark incubator until the start of the experiment 18-24 h later, when a small opaque rubber cap was glued over one eye. Exposure to the imprinting stimulus was begun 19-28 h post-hatch, which is later than in previous experiments. This delay was not expected seriously to impair imprinting since anaesthesia with sodium pentobarbitone is known to prolong the sensitive period 20. Immediately before exposure to the imprinting stimulus the chick was given an intracardiac injection of 20 /~Ci of tritiated uracil as described above. It was then placed in an activity wheel in front of a rotating flashing yellow light arranged so that the chick could turn the wheel freely, but stayed in a constant position 50 cm from the light. The apparatus and the light are described in detail elsewhere 7. The chick was trained for a total of 60 min with the light in 4 sessions of 15 min. Between successive intervals of training, the chick was kept in the dark for 15 min. This procedure was adopted since, in a pilot study, interspersed bouts of training were found to maintain chicks' responsiveness to the flashing light more effectively than a continuous period of exposure for 60 min (Bateson unpublished). After the last 15 min of training the chick was returned to the dark incubator for 20 min and then given a choice between the familiar flashing light and one which it had not seen before. After the test, which lasted 5 rain, the patch on the previously unexposed eye was removed and glued over the 'trained' eye. This operation took about 2 rain. Afterwards the chick was returned to the dark incubator for 13 rain. The chick was then given another choice test lasting 5 min. In both tests, which were intended to provide a measure of the strength of a chick's preference for the familiar yellow flashing light, the chick was given a choice between the familiar light and a rotating flashing red light. Full details of the method which is a form ofbehavioural titration
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are given elsewhere 7. In essence, the greater the positive score the stronger the preference for the familiar stimulus. The air temperature in the training apparatus was 29-31 °C, in the incubator it was 32.5-33.5 °C, and in the test situation it was 23-25 °C. A total of 36 chicks were trained and tested, but only 12 were used for biochemical analysis. In 6 of these the left eye was exposed to the yellow flashing light and in the other 6 the right eye. The criteria for selection were that the bird should show visual orientation with each eye and show no motor impairment. Most important of all, it had to show a marked preference for the familiar flashing yellow light on the exposed side and no clear preference on the other side. Biochemical analyses were not performed on chicks that failed to meet these criteria. Immediately after the final choice test and 150 min after injection, chicks which had met the criteria for biochemical analysis were decapitated. Their brains were dissected, frozen and analysed in the same way as in the first series of experiments except that specific activities were not corrected for body weight. All statistical tests referred to in the text were two-tailed unless otherwise stated. RESULTS
Intact chicks
In the first experiment with these chicks, each bird was trained with the flashing light for 76 min. The mean values and standard errors of the standardised specific activities for the trained and untrained sides of the brain (contralateral to the respective eye) are shown in Table I. There are no significant differences in any region between the two sides. When chicks with both eyes uncovered were exposed to a flashing light for 76 min the incorporation of tritiated uracil into presumed R N A was increased in the forebrain roof, compared with light and dark controls 5. The failure to find a difference between trained and untrained sides in the present experiments could be attributed to the chicks with a cap over one eye receiving less stimulation than chicks with both eyes uncovered. In that case neither side of the brain might have received stimulation sufficient to increase the amount of uracil incorporated into acidinsoluble substances. We therefore repeated the experiment, but increased the length TABLE I STANDARDISED SPECIFIC ACTIVITIES FOR RADIOACTIVE URACIL INCORPORATED INTO ACID-INSOLUBLE SUBSTANCES OF 3 BRAIN REGIONS OF INTACT CHICKS IN WHICH ONE EYE ONLY WAS EXPOSED TO THE IMPRINTING STIMULUS
Group
Side
Roof
Base
Midbrain
76 rain N ~ 12
Trained Untrained
100.2 4- 2.0 99.8 -4-2.0
100.9 4- 3.8 101.9 -4-2.5
102.1 + 2.7 96.0 -6 2.7
155 min N = 11
Trained Untrained
101.6 4- 3.2 97.4 4- 2.0
98.7 4- 2.2 96.8 -4- 1.2
102.2 4- 2.5 103.4 4- 2.8
Left side o f brain trained Midbrain Left Right Forebrain base Left Right Forebrain roof Left Right
Right side o f brain trained Midbrain Left Right Forebrain base Left Right Forebrain roof Left Right
552.1 526.7 504.6 461.3 533.5 539.3
7
Chick no.
504.9 437.0 457.0 367.7 424.2 462.5
1
Chick no.
458.8 434.7 392.0 401.2 375.3 398.9
8
690.4 636.6 603.9 594.3 295.1 662.1
2
433.2 360.5 369.0 368.0 437.9 361.9
9
394.6 402.3 353.0 308.6 392.6 412.6
3
425.2 385.6 329.4 359.4 371.7 339.5
10
379.7 342.0 224.5 344.1 347.6 362.1
4
318.0 270.7 208.5 190.7 219.7 216.5
11
369.2 351.5 319.0 297.4 282.4 368.4
5
308.7 310.0 331.1 271.4 287.7 278.2
12
299.5 308.3 265.3 239.3 185.4 266.9
6
± ± ± ± ± ±
2.0 2.7 5.7 3.7 8.6 1.8
113.8 103.8 96.4 91.7 99.5 95.2
2:4.4 L 2.8 _4- 3.4 -L 2.7 ~ 3.2 -~ 1.9
Standardisea means ± S.E.
113.0 107.0 94.4 92.0 85.2 108.4
Standardised means ~ S.E.
T h e u n s t a n d a r d i s e d values are expressed as d i s i n t . / m i n / m g protein. T o calculate the s t a n d a r d i s e d m e a n s , each value was expressed as a percentage o f the m e a n for all brain regions f r o m t h a t chick.
UNSTANDARDISEDAND STANDARDISEDSPECIFIC ACTIVITIES FOR RADIOACTIVEURACIL INCORPORATED INTO ACID-INSOLUBLESUBSTANCESOF 3 BRAIN REGIONS
T A B L E II
z
t,~ ta,a
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IMPRINTING IN 'SPLIT-BRAIN' CHICKS
of training to the flashing light to 155 min. The results of this experiment are also shown in Table I. Again no regional differences in the incorporation of tritiated uracil into presumed RNA were found between the trained and untrained sides of the brain. In view of the findings described below we compared the mean regional values of the standardised specific activities from the right side of the brain with those from the left side. No significant differences were found.
'Split-brain' chicks When the trained eye of each chick was exposed in the test, the mean score obtained by the chicks was 8.64 i 2.59; when the untrained eye was tested the score was 0.58 ± 1.00. The unstandardised data and the means and standard errors for the standardised specific activities of each brain region of each group are shown in Table II. The effect of training can be assessed statistically with a matched pairs t-test by calculating the differences between trained and untrained sides. The only significant difference occurred in the roof region of the forebrain where the trained side was 15.2 ~ higher than the untrained side (t = 2.74, P < 0.01, one-tailed). In addition to the effects of stimulation, incorporation in the two brain regions differed between left and right sides irrespective of which sides were trained. In the midbrain the incorporation of uracil into acid-insoluble material was significantly higher on the left side than on the right side (matched pairs t = 3.62, P < 0.01). In the roof the right side was higher than the left though the difference was not statistically significant (t ---- 1.88, 0.05 < P < 0.1). It can be seen in Table II that an interaction occurred between the effects of training and the left-right assymetry. In Table III standardised values for total radioactivity have been given. The difference between trained and untrained sides borders on significance in the midbrain (matched pairs t = 2.18, 0.05 < P < 0.1), otherwise the values are virtually identical for the two sides. No differences occurred between left and right sides in the total radioactivity values.
TABLE III STANDARDISED SPECIFIC ACTIVITIES FOR TOTAL POOLS OF RADIOACTIVE URACIL IN 'SPLIT-BRAIN' EXPERIMENTS
The 'trained' side of the brain is contralateral to the 'trained' eye.
Group
Side of brain
Roof
Base
Left-eye trained N = 6 Right-eye trained N = 6
Trained (right) Untrained (left) Trained (left) Untrained (right)
92.2 92.6 96.5 91.8
104.3 100.9 100.7 101.3
± 1.0 ± 2.3 q- 2.1 4- 1.4
Midbrain 4- 1.4 i 1.8 4- 2.3 4- 2.1
103.0 107.0 102.1 107.6
q- 2.9 4- 2.1 4- 0.6 =I: 4.1
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DISCUSSION
In the first series of experiments reported in this paper we examined the effects of a monocular imprinting procedure on presumed RNA synthesis in the brains of intact chicks. We failed to find a difference in any brain region. In view of the evidence for interhemispheric transfer in young ducklings and chicks 9,z4, the negative results are not surprising. In the second series of experiments using 'split-brain' chicks trained for 60 rain, incorporation into the trained side was significantly higher than into the untrained side in the forebrain roof region alone. This is the region in which we have previously detected differences between experimental and control chicks in the incorporation of lysine into presumed protein and where we have found the most rapid incorporation of uracil into presumed RNA as a result of exposure to an imprinting stimulus ~. In contrast, no significant regional differences in pool size were found between the two sides of the brain. This finding rules out the possibility that incorporation of the labelled base into macromolecules can be ascribed to asymmetric changes in pool size resulting, for example, from differences in cerebral blood flow which can be affected by patterned visual stimulation s . In the experiments of this paper, as with our earlier ones, we have studied the effect of a training procedure on a biochemical measure, the incorporation of labelled precursor into presumed RNA. The biochemical interpretations of this measure are limited in ways which we have discussed elsewhere 1~. Problems of the kinetics of uptake, the incorporation of tritium into molecules other than those being examined aT, of possible variations in pool size and of the distinction between incorporation of a precursor and net synthesis of a macromolecule all limit the weight to be placed upon observations made using this technique. For our present purposes these limitations are largely immaterial; our purpose is to relate a procedure which results in a behavioural change to changes in a biochemical index of neural activity using each animal as its own control. The precise nature of the biochemical differences between trained and untrained sides of the forebrain roof is irrelevant to determining whether or not these changes are specifically and exclusively related to the training procedure. Three previous biochemical studies have been reported in which each animal was used as its own control. In the first of these Hydrn and Egyhfizi1~ trained intact right-handed rats to obtain food by reaching with the left forepaw. They found an increase in neuronal RNA and a change in RNA base ratios in the forepaw region of the right sensorimotor cortex. This region is, of course, necessary for the control of movement of the left forepaw (el Peterson and DevineZS). It seems likely, therefore, that Hydrn's and Egyh~.zi's 15 results were due to increased use of the left forepaw -- a form of 'work hypertrophy' - - rather than to learning as such. Hydrn and Lange 16 found that both the incorporation of labelled leucine into presumed protein and the total pool of labelled leucine was higher on the side ipsilateral to the used paw than on the contralateral side. However, the ratio of incorporated leucine to total pool was higher on the contralateral side. This finding is not easy to interpret since the relationship between incorporation of leucine into acid-insoluble substances and total
IMPRINTING IN 'SPLIT-BRAIN' CHICKS
235
pool may not be strictly linear in the hippocampus. In our own work we have observed no obvious relationship between incorporation of labelled lysine and total pool in the chick brainL Metzger et al. ~1 used the 'split-brain' rhesus monkey (Macaca mulatta) as its own control. They studied the effects of monocular training on protein synthesis in the occipital and temporal cortex of both hemispheres and failed to find any differences between the two sides. No other regions were studied. Even if biochemical changes occur in the occipital and temporal cortical areas of monkeys after training, it is possible that the procedures used (for example, diffuse light flash as the conditioned stimulus and tritiated water as a precursor of protein) were not sufficiently sensitive to detect them. In the experiments on 'split-brain' chicks we found, unexpectedly, asymmetries in incorporation into the roof and midbrain regions, regardless of which side was trained. The difference between left and right sides was not statistically significant in the roof region. Any differences would be partly obscured by the effects of training. When the right side of the brain was stimulated the effects of training on uracil incorporation into the right forebrain roof would have been superimposed on the already higher rate of incorporation on that side. However, when the left forebrain roof was stimulated, the effects of training would have reduced the difference between the two sides. No differences between left and right sides of any brain region were found in the unoperated chicks. It may be relevant, therefore, that surgical manipulations were largely confined to the skull and dura above the left forebrain. If the operative interventions were responsible for the asymmetries the processes must be complex since incorporation into the left side tended to be lower than the right in the roof and was higher in the midbrain. We conclude that the effects of our imprinting procedure on the incorporation of uracil cannot be attributed to some of the general consequences of training. The 'splitbrain' preparation does not, however, eliminate all such non-specific effects (see Bateson3) and it remains possible that the biochemical differences are due to sensory stimulation as such or to an interaction between sensory stimulation and the hormonal state of the animal, and are not the exclusive effects of training. Although these possibilities are real, it is worth noting that the midbrain contains the large optic tectum which receives a massive input from the optic tract. However, the midbrain showed no changes in presumed RNA synthesis after 76 min of exposure to an imprinting stimulus, whereas changes were found in the forebrain roof ~ (see also Bateson et al.6a). ACKNOWLEDGEMENTS
We thank Mrs. A. L. D. Horn for help and discussion and Mr. A. K. Sinha for technical assistance. This work was supported by grants from the Science Research Council to G. H., the Medical Research Council to S.P.R.R., and the Agricultural and Science Research Councils to P.P.G.B.
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REFERENCES 1 ALTMAN, J., AND DAS, G. D., Behavioural manipulations and protein metabolism of the brain : effects of motor activity on the utilisation of leucine-H 3, Physiol. Behav., 1 (1966) 105-108. 2 AZM1TIA,E. G., JR., AND McEwEN, B. S., Corticosterone regulation of tryptophan hydroxylase in midbrain of the rat, Science, 166 (1969) 1274-1276. 3 BATESON,P. P. G., Are they really the products of learning? in G. HORN AND R. A. HINDE (Eds.), Short-term Changes in Neural Activity and Behaviour, Cambridge University Press, Cambridge, 1970, pp. 553-564. 4 BATESON,P. P. G., HORN, G., AND ROSE, S. P. R., Effects of an imprinting procedure on regional incorporation of tritiated lysine into protein of chick brain, Nature (Lond.), 223 (1969) 534-535. 5 BATESON,P. P. G., HORN, G., AND ROSE, S. P. R., Effects of early experience on regional incorporation of precursors into RNA and protein in the chick brain, Brain Research, 39 (1972) 449-465. 6 BATESON,P. P. G., AND REESE, E. P., The reinforcing properties of conspicuous stimuli in the imprinting situation, Anita. Behav., 17 (1969) 692-699. 6a BATESON,P. P. G., RosE, S. P. R., AND HORN, G., Lasting effects of imprinting on uracil incorporation into chick brain, Science, in press. 7 BATESON,P. P. G., AND WAINWRIGHT,A. A. P., The effects of prior exposure to light on the imprinting process in domestic chicks, Behaviour, 42 (1972) 279-290. 8 [~NDY, S. C., AND MOREEOS,B. S., Stimulus deprivation and cerebral blood flow, Exp. Neurol., 31 (1971) 200-206. 9 CHERKIN,A., Eye to eye transfer of an early response modification in chicks, Nature (Lond.), 227 (1970) 1153. 10 COWAN, W. M., ADAMSON,L., AND POWELL, T. P. S., An experimental study of the avian visual system, J. Anat. (Lond.), 95 (1961) 545-563. 11 CU~NOD, M., ET ZEIER, H., Transfert visuel interh6misphOriques et commissurotomie chez le pigeon, Schweiz. Arch. Neurol. Neurochir. Psychiat., 100 (1967) 365-380. 12 HORN, G., HORN, A. L. D., BATESON,P. P. G., AND ROSE, S. P. R., Effects of imprinting on uracil incorporation into brain R N A in the 'split-brain' chick, Nature (Lond.), 229 (1971) 131-132. 13 HORN, G., BATESON,P. P. G., AND ROSE, S. P. R., Experience and plasticity in the central nervous system, Science, in press. 14 HYD~N, H., Protein metabolism in the nerve cell during growth and function, Acta physiol. scand., 6, Suppl. 17 (1943) 1-136. 15 HYDI~N,H., AND EGYH.~ZI, E., Changes in RNA content and base composition in cortical neurons of rats in a learning experiment involving transfer of handedness, Proc. nat. Acad. Sci. (Wash.), 52 (1964) 1030-1035. 16 HYDI~N, H., AND LANGE, P. W., Protein synthesis in the hippocampal pyramidal cells of rats during a behavioral test, Science, 159 (1968) 1370-1373. 17 JAKOUBEK,B., BURE~OVA,M., H.~JEK, I., ETRYCHOVA,J., PAVLIK,A., AND D~DI~OVA,A., Effect of ACTH on the synthesis of rapidly labelled R N A in the nervous system of mice, Brain Research, 43 (1972) 417-428. 18 JAKOUaEK, B., HOR.~KOV,~, M., AND GUTMAN, E., Changes of protein metabolism during increased functional activity in spinal motoneurones and sciatic nerve, Proc. Int. UnionPhysiol. Sci., 7 (1968) 215. 19 JAKOUBEK,B., SEMIGINOVSKY,B., AND DI~DI~OV.~,A., The effect of ACTH on the synthesis of proteins in spinal motoneurones as studied by autoradiography, Brain Research, 25 (1971) 133 141. 20 McDONALD, G., Imprinting: drug produced isolation and the sensitive period, Nature (Lond.), 217 (1968) 1158-1159. 21 MEIER, R. E., Interhemisph~irischer Transfer visueller Zweifachwahlen bei kommissurotomierten Tauben, Psychol. Forsch., 34 (1971) 220-245. 22 MEIER, R. E., AND CUI~NOD, M., A visual function of the supraoptic decussation in the pigeon, Experientia (Basel), 26 (1970) 682. 23 METZGER, H. P., CUI~NOD, M., GRYNRAUM,A., AND WAELSCH, H., The effect of unilateral stimulation on synthesis of cortical proteins in each hemisphere of the split-brain monkey, J. Neurochem., 14 (1967) 183-187. 24 MOLTZ, H., AND STETTNER, L. J., Interocular mediation of the following response after patterned light deprivation, J. comp. physiol. Psychol., 55 (1962) 626-632.
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25 PETERSON, G. M., AND DEVINE, J. V., Transfer in handedness in the rat resulting from small cortical lesions after limited forced practice, J. comp. physiol. Psychol., 56 (1963) 752-756. 26 ROSE, S. P. R., BATESON,P. P. G., HORN, G., AND HORN, A. L. D., Effects of an imprinting procedure on regional incorporation of tritiated uracil into chick brain RNA, Nature (Lond.), 225 (1970) 650-651. 27 SEMIGINOVSKY,B., JAKOUBEK,B., KRAUSS, M., AND ERD~3SSOVX,R., Effects of restraint stress on the oxygen consumption and [14C]leucine incorporation into the brain cortex slices in rats, Brain Research, 23 (1970) 298-301.
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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
MONOCULAR IMPRINTING AND REGIONAL INCORPORATION OF TRITIATED URACIL INTO THE BRAINS OF INTACT AND 'SPLITBRAIN' CHICKS
G. H O R N , S. P. R. ROSE AND P. P. G. BATESON
University of Cambridge, Department of Anatomy, Cambridge, Department of Biology, Open University, Bletchley, Bucks. and University of Cambridge, Sub-Department of Animal Behaviour, Madingley, Cambridge (Great Britain) (Accepted December 6th, 1972)
SUMMARY
When intact chicks with a patch over one eye were exposed at a post-hatch age of 14-19 h to a flashing light, no regional differences between the two hemispheres were found in the incorporation of tritiated uracil into acid-insoluble residues. Similar biochemical studies were made using 12 chicks in which the supraoptic commissure had been transected. Each chick had one eye covered with a patch and was exposed to a flashing yellow light for a total of 60 min. It was subsequently given two choice tests between the familiar flashing yellow light and an unfamiliar flashing red light, first with its trained eye alone exposed and then with its untrained eye alone exposed. All of the 12 chicks approached the familiar flashing yellow light with the originally trained eye uncovered, but not with the originally untrained eye uncovered. The rate of incorporation of tritiated uracil into presumed RNA was 15.2 ~ higher in the trained side of the forebrain roof than in the untrained side. No other regional differences between trained and untrained sides were observed. The results show that certain non-specific consequences of the imprinting procedure, such as general changes in hormonal levels as a result of stress, non-visual sensory stimulation and motor activity, cannot account for the observed biochemical changes in the forebrain roof.
INTRODUCTION
When newly hatched domestic chicks are exposed to a conspicuous object they rapidly learn its characteristics and subsequently may approach it in preference to
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dissimilar objects. The learning process by which young birds form a social attachment for an object to which they have been exposed is called 'imprinting'. We have tound that when domestic chicks are exposed to a highly-effective imprinting stimulus, a rotating flashing light, changes occur in the presumed rates of protein and RNA synthesis in the brain4.5, 26. Of special interest was the discovery that after 76 min of exposure a sharp increase in the incorporation of tritiated uracil into acid-insoluble substances occurred in the upper part of the forebrain alone. Such effects were not found in chicks exposed to a flashing light for a shorter period of time, in a control group kept in the dark, or in chicks exposed to a steady level of background illumination for the same length of time. A number of factors could account for the biochemical differences between the experimental group exposed for 76 min and the other chicks. The groups may, for example, have differed in the amount of motor activity and in the levels of stress to which they were variously subjected. Protein and RNA metabolism in the central nervous system may be affected by motor activity xa,ls, by stress 1,27, and by exogenous A C T H 17A9 or corticosteroneL The experiments using exogenous hormones suggest that any procedure which modifies the endogenous levels of these substances may have repercussions on RNA and protein synthesis in the brain. Such general effects, which may often accompany learning, can largely be allowed for by effectively restricting input to one side of the brain during the training procedures. If any biochemical differences exist between the 'trained' and 'untrained' sides of the brain, it is reasonable to ascribe them to differences in visual experience. They are unlikely to result solely from general changes in hormonal levels as a consequence of stress, to differences in non-visual sensory stimulation, or to differences in motor activity between the corresponding sides of the body. Interhemispheric transfer takes place when only one eye is exposed during the imprinting of intact Peking ducklings 24. Similarly, a pilot study indicated that after exposure to a flashing light, day-old domestic chicks with one eye occluded showed a preference for that light when the untrained eye was subsequently tested (Bateson unpublished). Moreover, transfer of an acquired aversion to pecking at objects treated with methyl anthranilate has been observed in young chicks 9. All these studies suggest that both sides of the brain of the intact chick are trained when input is restricted to one side. Therefore, in the first series of experiments described here, we studied intact chicks which had one eye occluded. No differences between stimulated and unstimulated sides were found and so, in the second series, attempts were made to interrupt transfer from one side of the brain to the other. Transfer of a visual discrimination task is severely impaired in pigeons, the optic chiasma of which is completely crossed 1°, following transection of the supraoptic commissureZl,2L It seemed reasonable, therefore, to suppose that this surgical procedure would impair interhemispheric transfer in chicks which had one eye occluded when exposed to an imprinting stimulus. In the second series of experiments described here the incorporation of tritiated uracil into presumed RNA was studied in 'split-brain' chicks which had had only one eye exposed during the imprinting procedure. A preliminary account of the second series of experiments has already been publishedtL
IMPRINTING IN 'SPLIT-BRAIN' CHICKS
229
MATERIALS AND METHODS
Intact chicks The first series of experiments were conducted on unoperated Chunky chicks which were hatched and reared in the dark. Each chick had an opaque rubber cap (12 mm in diameter × 3 mm deep) fitted over one eye. The cap was attached round the eye with a rubber-based false eye-lash adhesive ('Eyelure'). Subsequently the chicks were marked, weighed, and, between 14-19 h after hatching, given an intracardiac injection of 20 #Ci [5-3H]uracil (specific activity 1000 mCi/mmole, Radiochemical Centre, Amersham, Bucks.) in 0.1 ml of Locke solution. Handling of the chicks was kept to a minimum and was always done in the dark or with the aid of a dim green light. Each chick was killed by decapitation 150 min after it had been injected. In the first experiment 12 chicks from the same hatch were used. Half had a cap fitted over their left eye and half over the right eye. After injection each chick was placed for 37 min in a dark incubator kept at 29-30 °C. It was then transferred to a training pen and exposed to an orange flashing light for 76 min. Details of the apparatus and methods are given elsewhere 5. After exposure the chicks were returned to the dark incubator for 35 min, given a 2 rain test and then killed. In the second experiment of this series, 11 chicks from one hatch were treated in a similar way but were exposed for 155 min to the flashing light. The period of exposure started 5 min before injection. In this experiment 6 chicks had the right eye exposed and 5 the left eye; originally the number was 6 but one chick lost its patch during the experiment and was discarded. As in our previous studies, the brain was dissected into forebrain roof, forebrain base and midbrain. The 3 regions were then divided down the median plane. The regions were placed into coded specimen pots and rapidly frozen on solid CO2. They were kept frozen until biochemical analyses were performed on each sample. The codes were not known by those performing the analyses. The samples were treated as before 5. Specific activities, expressed as disint./min/mg protein in the sample of the acid-insoluble fraction varied considerably between chicks. Values for all 6 samples from a brain were therefore standardised as percentages of the mean specific activity for that brain. As protein values obtained for any region did not vary significantly with behavioural treatment, expressing the results in this way was taken as a measure of incorporation into presumed RNA. Total radioactivity (pool) consisting of radioactivity in the acid-soluble plus the acid-insoluble fractions was measured and standardised in the same way. To assess the validity of the measures of specific activity used, the time-course of uptake and incorporation of [all]uracil into the brains of day-old chicks was studied in an earlier experiment. Each chick received an intracardiac injection of 20 #Ci [3H]uracil and they were killed at varying times afterwards. Uptake, as measured by changes in total pool, increased linearly for the first 60 min and then levelled off, remaining constant for at least a further 90 min. Incorporation into acid-insoluble substances proceeded linearly for at least 150 min, the activity of acid-insoluble relative to total pool at the end of this time being less than 6 ~ .
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"Split-brain' chicks In the second series of experiments Chunky chicks were hatched and kept in a dark incubator for 1--4 h after hatching. Each chick was weighed and anaesthetized by an intraperitoneal injection of 0.1 ml of 'Equithesin'. The cartilaginous skull was incised in a parasagittal plane approximately 3 mm to the left of the midline. A flap of cartilage extending to approximately 1 mm beyond the midline on the right was separated from the underlying dura and folded down. The dura overlying the left hemisphere was incised and reflected to expose the interhemispheric fissure. Following a technique similar to that described by Cu6nod and Zeier 11 a fine knife was lowered into the fissure, damage to the sagittal sinus being avoided by applying gentle traction on the dural flap. The blade was lowered to the best setting for the supraoptic commissure. This setting was determined by direct observation of the position of this commissure in a preliminary series of experiments and was found to be 2.0 mm anterior and 2.5 mm above the interaural plane in chicks weighing 35-50 g. These coordinates refer to a chick which is held in the stereotaxic apparatus with the upper surface of the beak bar, inserted between upper and lower beaks, set 2.7 mm below the interaural plane. After positioning, the knife was moved approximately 3 mm in the sagittal plane and slowly withdrawn. The dural and cartilaginous flaps were replaced and the skin wound closed. The surgical procedures were conducted using an operating microscope. Atter operation, the chick was maintained in a dark incubator until the start of the experiment 18-24 h later, when a small opaque rubber cap was glued over one eye. Exposure to the imprinting stimulus was begun 19-28 h post-hatch, which is later than in previous experiments. This delay was not expected seriously to impair imprinting since anaesthesia with sodium pentobarbitone is known to prolong the sensitive period 20. Immediately before exposure to the imprinting stimulus the chick was given an intracardiac injection of 20 /~Ci of tritiated uracil as described above. It was then placed in an activity wheel in front of a rotating flashing yellow light arranged so that the chick could turn the wheel freely, but stayed in a constant position 50 cm from the light. The apparatus and the light are described in detail elsewhere 7. The chick was trained for a total of 60 min with the light in 4 sessions of 15 min. Between successive intervals of training, the chick was kept in the dark for 15 min. This procedure was adopted since, in a pilot study, interspersed bouts of training were found to maintain chicks' responsiveness to the flashing light more effectively than a continuous period of exposure for 60 min (Bateson unpublished). After the last 15 min of training the chick was returned to the dark incubator for 20 min and then given a choice between the familiar flashing light and one which it had not seen before. After the test, which lasted 5 rain, the patch on the previously unexposed eye was removed and glued over the 'trained' eye. This operation took about 2 rain. Afterwards the chick was returned to the dark incubator for 13 rain. The chick was then given another choice test lasting 5 min. In both tests, which were intended to provide a measure of the strength of a chick's preference for the familiar yellow flashing light, the chick was given a choice between the familiar light and a rotating flashing red light. Full details of the method which is a form ofbehavioural titration
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IMPRINTING IN 'SPLIT-BRAIN' CHICKS
are given elsewhere 7. In essence, the greater the positive score the stronger the preference for the familiar stimulus. The air temperature in the training apparatus was 29-31 °C, in the incubator it was 32.5-33.5 °C, and in the test situation it was 23-25 °C. A total of 36 chicks were trained and tested, but only 12 were used for biochemical analysis. In 6 of these the left eye was exposed to the yellow flashing light and in the other 6 the right eye. The criteria for selection were that the bird should show visual orientation with each eye and show no motor impairment. Most important of all, it had to show a marked preference for the familiar flashing yellow light on the exposed side and no clear preference on the other side. Biochemical analyses were not performed on chicks that failed to meet these criteria. Immediately after the final choice test and 150 min after injection, chicks which had met the criteria for biochemical analysis were decapitated. Their brains were dissected, frozen and analysed in the same way as in the first series of experiments except that specific activities were not corrected for body weight. All statistical tests referred to in the text were two-tailed unless otherwise stated. RESULTS
Intact chicks
In the first experiment with these chicks, each bird was trained with the flashing light for 76 min. The mean values and standard errors of the standardised specific activities for the trained and untrained sides of the brain (contralateral to the respective eye) are shown in Table I. There are no significant differences in any region between the two sides. When chicks with both eyes uncovered were exposed to a flashing light for 76 min the incorporation of tritiated uracil into presumed R N A was increased in the forebrain roof, compared with light and dark controls 5. The failure to find a difference between trained and untrained sides in the present experiments could be attributed to the chicks with a cap over one eye receiving less stimulation than chicks with both eyes uncovered. In that case neither side of the brain might have received stimulation sufficient to increase the amount of uracil incorporated into acidinsoluble substances. We therefore repeated the experiment, but increased the length TABLE I STANDARDISED SPECIFIC ACTIVITIES FOR RADIOACTIVE URACIL INCORPORATED INTO ACID-INSOLUBLE SUBSTANCES OF 3 BRAIN REGIONS OF INTACT CHICKS IN WHICH ONE EYE ONLY WAS EXPOSED TO THE IMPRINTING STIMULUS
Group
Side
Roof
Base
Midbrain
76 rain N ~ 12
Trained Untrained
100.2 4- 2.0 99.8 -4-2.0
100.9 4- 3.8 101.9 -4-2.5
102.1 + 2.7 96.0 -6 2.7
155 min N = 11
Trained Untrained
101.6 4- 3.2 97.4 4- 2.0
98.7 4- 2.2 96.8 -4- 1.2
102.2 4- 2.5 103.4 4- 2.8
Left side o f brain trained Midbrain Left Right Forebrain base Left Right Forebrain roof Left Right
Right side o f brain trained Midbrain Left Right Forebrain base Left Right Forebrain roof Left Right
552.1 526.7 504.6 461.3 533.5 539.3
7
Chick no.
504.9 437.0 457.0 367.7 424.2 462.5
1
Chick no.
458.8 434.7 392.0 401.2 375.3 398.9
8
690.4 636.6 603.9 594.3 295.1 662.1
2
433.2 360.5 369.0 368.0 437.9 361.9
9
394.6 402.3 353.0 308.6 392.6 412.6
3
425.2 385.6 329.4 359.4 371.7 339.5
10
379.7 342.0 224.5 344.1 347.6 362.1
4
318.0 270.7 208.5 190.7 219.7 216.5
11
369.2 351.5 319.0 297.4 282.4 368.4
5
308.7 310.0 331.1 271.4 287.7 278.2
12
299.5 308.3 265.3 239.3 185.4 266.9
6
± ± ± ± ± ±
2.0 2.7 5.7 3.7 8.6 1.8
113.8 103.8 96.4 91.7 99.5 95.2
2:4.4 L 2.8 _4- 3.4 -L 2.7 ~ 3.2 -~ 1.9
Standardisea means ± S.E.
113.0 107.0 94.4 92.0 85.2 108.4
Standardised means ~ S.E.
T h e u n s t a n d a r d i s e d values are expressed as d i s i n t . / m i n / m g protein. T o calculate the s t a n d a r d i s e d m e a n s , each value was expressed as a percentage o f the m e a n for all brain regions f r o m t h a t chick.
UNSTANDARDISEDAND STANDARDISEDSPECIFIC ACTIVITIES FOR RADIOACTIVEURACIL INCORPORATED INTO ACID-INSOLUBLESUBSTANCESOF 3 BRAIN REGIONS
T A B L E II
z
t,~ ta,a
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IMPRINTING IN 'SPLIT-BRAIN' CHICKS
of training to the flashing light to 155 min. The results of this experiment are also shown in Table I. Again no regional differences in the incorporation of tritiated uracil into presumed RNA were found between the trained and untrained sides of the brain. In view of the findings described below we compared the mean regional values of the standardised specific activities from the right side of the brain with those from the left side. No significant differences were found.
'Split-brain' chicks When the trained eye of each chick was exposed in the test, the mean score obtained by the chicks was 8.64 i 2.59; when the untrained eye was tested the score was 0.58 ± 1.00. The unstandardised data and the means and standard errors for the standardised specific activities of each brain region of each group are shown in Table II. The effect of training can be assessed statistically with a matched pairs t-test by calculating the differences between trained and untrained sides. The only significant difference occurred in the roof region of the forebrain where the trained side was 15.2 ~ higher than the untrained side (t = 2.74, P < 0.01, one-tailed). In addition to the effects of stimulation, incorporation in the two brain regions differed between left and right sides irrespective of which sides were trained. In the midbrain the incorporation of uracil into acid-insoluble material was significantly higher on the left side than on the right side (matched pairs t = 3.62, P < 0.01). In the roof the right side was higher than the left though the difference was not statistically significant (t ---- 1.88, 0.05 < P < 0.1). It can be seen in Table II that an interaction occurred between the effects of training and the left-right assymetry. In Table III standardised values for total radioactivity have been given. The difference between trained and untrained sides borders on significance in the midbrain (matched pairs t = 2.18, 0.05 < P < 0.1), otherwise the values are virtually identical for the two sides. No differences occurred between left and right sides in the total radioactivity values.
TABLE III STANDARDISED SPECIFIC ACTIVITIES FOR TOTAL POOLS OF RADIOACTIVE URACIL IN 'SPLIT-BRAIN' EXPERIMENTS
The 'trained' side of the brain is contralateral to the 'trained' eye.
Group
Side of brain
Roof
Base
Left-eye trained N = 6 Right-eye trained N = 6
Trained (right) Untrained (left) Trained (left) Untrained (right)
92.2 92.6 96.5 91.8
104.3 100.9 100.7 101.3
± 1.0 ± 2.3 q- 2.1 4- 1.4
Midbrain 4- 1.4 i 1.8 4- 2.3 4- 2.1
103.0 107.0 102.1 107.6
q- 2.9 4- 2.1 4- 0.6 =I: 4.1
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G. HORN el al.
DISCUSSION
In the first series of experiments reported in this paper we examined the effects of a monocular imprinting procedure on presumed RNA synthesis in the brains of intact chicks. We failed to find a difference in any brain region. In view of the evidence for interhemispheric transfer in young ducklings and chicks 9,z4, the negative results are not surprising. In the second series of experiments using 'split-brain' chicks trained for 60 rain, incorporation into the trained side was significantly higher than into the untrained side in the forebrain roof region alone. This is the region in which we have previously detected differences between experimental and control chicks in the incorporation of lysine into presumed protein and where we have found the most rapid incorporation of uracil into presumed RNA as a result of exposure to an imprinting stimulus ~. In contrast, no significant regional differences in pool size were found between the two sides of the brain. This finding rules out the possibility that incorporation of the labelled base into macromolecules can be ascribed to asymmetric changes in pool size resulting, for example, from differences in cerebral blood flow which can be affected by patterned visual stimulation s . In the experiments of this paper, as with our earlier ones, we have studied the effect of a training procedure on a biochemical measure, the incorporation of labelled precursor into presumed RNA. The biochemical interpretations of this measure are limited in ways which we have discussed elsewhere 1~. Problems of the kinetics of uptake, the incorporation of tritium into molecules other than those being examined aT, of possible variations in pool size and of the distinction between incorporation of a precursor and net synthesis of a macromolecule all limit the weight to be placed upon observations made using this technique. For our present purposes these limitations are largely immaterial; our purpose is to relate a procedure which results in a behavioural change to changes in a biochemical index of neural activity using each animal as its own control. The precise nature of the biochemical differences between trained and untrained sides of the forebrain roof is irrelevant to determining whether or not these changes are specifically and exclusively related to the training procedure. Three previous biochemical studies have been reported in which each animal was used as its own control. In the first of these Hydrn and Egyhfizi1~ trained intact right-handed rats to obtain food by reaching with the left forepaw. They found an increase in neuronal RNA and a change in RNA base ratios in the forepaw region of the right sensorimotor cortex. This region is, of course, necessary for the control of movement of the left forepaw (el Peterson and DevineZS). It seems likely, therefore, that Hydrn's and Egyh~.zi's 15 results were due to increased use of the left forepaw -- a form of 'work hypertrophy' - - rather than to learning as such. Hydrn and Lange 16 found that both the incorporation of labelled leucine into presumed protein and the total pool of labelled leucine was higher on the side ipsilateral to the used paw than on the contralateral side. However, the ratio of incorporated leucine to total pool was higher on the contralateral side. This finding is not easy to interpret since the relationship between incorporation of leucine into acid-insoluble substances and total
IMPRINTING IN 'SPLIT-BRAIN' CHICKS
235
pool may not be strictly linear in the hippocampus. In our own work we have observed no obvious relationship between incorporation of labelled lysine and total pool in the chick brainL Metzger et al. ~1 used the 'split-brain' rhesus monkey (Macaca mulatta) as its own control. They studied the effects of monocular training on protein synthesis in the occipital and temporal cortex of both hemispheres and failed to find any differences between the two sides. No other regions were studied. Even if biochemical changes occur in the occipital and temporal cortical areas of monkeys after training, it is possible that the procedures used (for example, diffuse light flash as the conditioned stimulus and tritiated water as a precursor of protein) were not sufficiently sensitive to detect them. In the experiments on 'split-brain' chicks we found, unexpectedly, asymmetries in incorporation into the roof and midbrain regions, regardless of which side was trained. The difference between left and right sides was not statistically significant in the roof region. Any differences would be partly obscured by the effects of training. When the right side of the brain was stimulated the effects of training on uracil incorporation into the right forebrain roof would have been superimposed on the already higher rate of incorporation on that side. However, when the left forebrain roof was stimulated, the effects of training would have reduced the difference between the two sides. No differences between left and right sides of any brain region were found in the unoperated chicks. It may be relevant, therefore, that surgical manipulations were largely confined to the skull and dura above the left forebrain. If the operative interventions were responsible for the asymmetries the processes must be complex since incorporation into the left side tended to be lower than the right in the roof and was higher in the midbrain. We conclude that the effects of our imprinting procedure on the incorporation of uracil cannot be attributed to some of the general consequences of training. The 'splitbrain' preparation does not, however, eliminate all such non-specific effects (see Bateson3) and it remains possible that the biochemical differences are due to sensory stimulation as such or to an interaction between sensory stimulation and the hormonal state of the animal, and are not the exclusive effects of training. Although these possibilities are real, it is worth noting that the midbrain contains the large optic tectum which receives a massive input from the optic tract. However, the midbrain showed no changes in presumed RNA synthesis after 76 min of exposure to an imprinting stimulus, whereas changes were found in the forebrain roof ~ (see also Bateson et al.6a). ACKNOWLEDGEMENTS
We thank Mrs. A. L. D. Horn for help and discussion and Mr. A. K. Sinha for technical assistance. This work was supported by grants from the Science Research Council to G. H., the Medical Research Council to S.P.R.R., and the Agricultural and Science Research Councils to P.P.G.B.
236
~. HORN el al.
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