Imprinting: Correlations between behaviour and incorporation of [14C]uracil into chick brain

Brain Research, 84 (1975) 207-220

207

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

IMPRINTING: CORRELATIONS BETWEEN BEHAVIOUR AND INCORPORATION OF [14C]URACIL INTO CHICK BRAIN

P. P. G. BATESON, G. H O R N ANt) S. P. R. ROSE

University of Cambridge, Sub-Department of Animal Behaviour, Madingley, Cambridge; Department of Anatomy, The University, Bristol and Brain Research Group, Open University, Bletchley, Bucks. (Great Britain) (Accepted September 22nd, 1974)

SUMMARY

On the first day after hatching 106 domestic chicks were injected with [14C]uracil and exposed to a yellow or a red rotating flashing light for 72 min. Shortly before they were killed and 115 rain after injection they were given a choice between the two lights. Incorporation of the [14C]uracil into 4 regions of the brain was measured. Differences between batches and between training conditions were eliminated as sources of variation by standardising the behavioural and biochemical measures. Correlations between the measures were obtained. Chicks that were active from the early stages of training showed lower rates of incorporation in the midbrain, forebrain base and anterior roof of the forebrain, than less responsive chicks. When the general lowering effect of behavioural activity on incorporation was eliminated as a source of variation, a striking positive correlation emerged between the preference for the familiar object and incorporation in the anterior forebrain roof. Since preference for the familiar is a direct measure of imprinting and since no other behavioural measure is positively correlated with biochemical changes in anterior roof, it is concluded that these biochemical changes were closely linked with the learning process.

INTRODUCTION

Day-old chicks previously reared in the dark readily approach a variety of visually conspicuous objects. During the period of exposure to an object, their preferences are increasingly restricted to that objecta,17,27. The learning process involved in the establishment of a specific social attachment is commonly called 'imprinting'. After day-old domestic chicks have been exposed to a flashing rotating light, which is a highly effective imprinting stimulus7, a variety of localised biochemical changes have been found in the brain. In particular, within 30 min of the onset of exposure, the

208 activity of the enzyme RNA polymerase was elevated in the forebrain roof region relative to birds kept in the darklL The incorporation of [ZH]uracil into presumed RNA was greater after the chicks had been exposed to the flashing light for 76 rain, and following 1 h or more og exposure, [3H]- and [x4C]lysine incorporation into presumed protein in the same region was also greater than in birds kept in the dark '~,~. Comparable differences between trained and untrained birds were not found in the forebrain base and the midbrain. However, after being exposed for 160 rain either to a flashing rotating light or to a continuous overhead light similar differences between the birds exposed to light and those kept in the dark were found in all brain regions when [ZH]uracil was used as precursor 9. These widespread changes were probably due to general consequences of prolonged sensory stimulation and so, in our subsequent work, we have concentrated on analysing the factors responsible for the rapidly occurring changes localised in the forebrain roof. In particular, we have attempted to discover whether these changes can be attributed to neural events that are necessary for and exclusively related to the imprinting process. The rapidly occurring changes in the forebrain roof might have resulted from a variety of relatively non-specific factors such as motor activity, short-term effects of sensory stimulation or changes in hormonal level. Two series of experiments have suggested that such possibilities are unlikely. Incorporation of [ZH]uracil into presumed RNA was found to be higher in the forebrain roof of the 'trained' side of 'splitbrain' chicks than in the 'untrained' side 18. This result appeared to rule out the possibility that increased incorporation was simply produced by motor activity or changes in hormonal level. In the second series of experiments, chicks were trained for varying periods of time from 20 rain to 240 min on the first day after hatching, and for a further 60 min on the second day. Measurement of [3H]uracil incorporation was made on the second day, when all birds received the same period of sensory stimulation. If incorporation into a particular region of the brain were specifically related to learning, a lower rate should be found in birds that had previously been exposed for a longer period and had learned more of the characteristics of the stimulus object. This hypothesis assumes that the extent to which further learning takes place on the second day diminishes as the length of training is increased on the first day. We found that as the length of exposure was increased on the previous day so incorporation into the anterior part of the forebrain roof decreased 10. Since no such result was found in any other regions of the brain, we concluded that the rapidly occurring changes in the forebrain roof do not result from short-term sensory stimulation. The evidence we have accumulated suggests that the biochemical changes are necessary for imprinting, although our results could still be explained in terms of sideeffects of the training procedure interacting in complex ways to accelerate, for example, general neuronal development in the forebrain roof. Fortunately, the suggested close link with the imprinting process can be examined in quite different ways from those we have used in the past by exploiting the natural variation in the behaviour and brain biochemistry of birds. In the study reported in this paper we have used this approach and looked for associations between behavioural and biochemical measures from chicks which were trained for the same length of time and tested in the same way.

209 MATERIALS AND METHODS

Subjects One hundred and six domestic chicks were used, hatched from 10 separate batches of eggs. Fertile eggs of a broiler strain (Ross Chunkies from Ross Poultry Ltd., Fornham All Saints, Suffolk) were placed in an automatic incubator for 17-18 days after which they were transferred to a dark still-air incubator where they hatched. The chicks were transferred, generally within 3 h of hatching, to another dark incubator and kept there in individual compartments until the beginning of the experiment. 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 to which chicks are relatively unresponsive 23. The experiment was begun 12-24 h after hatching (mean = 20.85 h).

Training apparatus The apparatus used to train and test the chicks is described in detail elsewhere 7,s and is outlined only briefly here. The light source for each of the two stimuli used was a 12 V, 45 W lamp. A yellow filter was placed over one and a red filter over the other. A rectangular plastic box with the long sides translucent and the short sides opaque rotated about the yellow light at 65 rev./min. A translucent plastic cylinder painted with two diagonal black stripes between which were two black circular patches, rotated at the same rate about the red light. The chicks were trained in a running wheel 30 cm in diameter and 13 cm wide. The wheel could turn freely on a stand placed 50 cm from the rotating stimulus. The sides of each wheel were made of hardboard painted matt black. The curved surface on which the chick walked was made of wire mesh with a trap-door in it through which the chicks could be put into the wheel. As the chick moved toward the stimulus, the wheel rotated on the fixed stand and the chick remained on the bottom of the wheel Four pulses were generated for each revolution of the wheel in one direction through closure of a reed relay operated by 4 magnets spaced evenly and symmetrically on the side of each wheel. Forward and backward rotations of the wheel were automatically recorded in independent channels of a computer based event recorder 3°. The chicks were trained in a cabinet containing two running wheels and one light. All the inside surfaces of the cabinet were painted matt black and no illumination was provided apart from the rotating light. The cabinets were maintained at 30 °C at the chicks' positions with a maximum variation of 4- 0.2 °C.

Testing apparatus The testing apparatus consisted of a running wheel, similar to the one described above, mounted on a wheeled trolley which ran on two rails 250 cm in length. The yellow flashing light was placed at one end of the rails and the red light at the other end. These stimuli were identical to those used during training. The positions of the light were reversed after 5 batches had been tested. A mirror was placed at an angle above the apparatus so that it was possible to watch a chick while it was being tested. No illumination was provided for the test apparatus apart from the flashing rotating

210 lights. The temperature was kept at 23.5-26.5 ~:'C at the position of the chick. The running wheel and trolley were geared together in such a way that when the chick walked in the direction of, say the yellow flashing light, the trolley moved in the opposite direction and the chick, attempting to approach the yellow light, was carried away from it toward the red light. The distance moved by the trolley was read off a centimetre scale marked along a rail. The rationale for using this method of testing chicks' preferences in a choice situation has been given elsewhere4,5, 7. Essentially, if the chick was allowed to approach the stimulus toward which it first moved, the stimulus would subtend a progressively larger visual angle and would become progressively more attractive. Consequently no further information would be obtained about the extent of the chick's preference for the object it had approached over the other object used in the choice test. This difficulty can be overcome if the chick is carried away from the stimulus it is trying to approach, since the stimulus becomes progressively less conspicuous and hence less attractive to the chick.

Experimentalprocedure At the beginning of the experiment chicks were weighed and at 10-min intervals injected with 0.1 ml Locke solution containing 4 / , C i [2-14C]uracil (specific activity 60 mCi/mmole, from the Radiochemical Centre, Amersham, Bucks.) into the heart region. Immediately afterwards (see Fig. 1) each chick was placed in an individual compartment in a dark incubator maintained at 29.5-30.5 °C. The chick was then taken from the incubator, placed in one of the running wheels and its training stimulus was turned on exactly 15 min after injection. In the first 5 batches the first chick was trained with a yellow light, the second with a red light, the third with a yellow light and so on. In the second 5 batches the position of the light was altered and the order reversed. Altogether, after some deaths from the injection procedure, 54 chicks were trained with the yellow light and 52 with the red light. Each chick was left in its training wheel for a total of 80 min. The period was divided up into 8 sessions of 10 min during each of which the training stimulus was on for 9 min and off for the tenth rain. This method of interrupted exposure has been found to be a more effective method of training chicks than continuous exposure. The flashing lights were always off when the chicks were put into or withdrawn from their wheels. When a chick was taken from its wheel it was replaced in an individual compartment in the dark incubator kept at 29.5-30.5 °C. Twenty-one min after the end of training and

INJECT

6

KILL

20

4'0

(~0

Time (Min)

80

l(JO

120

Fig. 1. Plan of the experiment to show how the chicks were treated between injection with [z4C]uracil and killing 120 rain later.

211 TABLE I SUMMARY OF BEHAVIOURAL MEASURES

Training approach latency Initial approach Total approach Test latency Peep calls Test movement Preference for familiar

Time from beginning of training to first approach movement Amount of approach movements during training in 20 rain after first approach movement Total amount of approach movements throughout training period Time from beginning of test to first attempted approach movement Number of 'distress' calls given in first minute of test Total distance travelled by test wheel during choice test Greatest overall excursion of test wheel from midpoint on the rails during choice test. The greater the excursion away from the familiar object the stronger the preference for the familiar

115 min after it had been injected, each chick was given a choice in the testing apparatus between the familiar rotating light with which it had been trained and the other rotating light. Before the test was started the wheel was placed exactly midway between the ends of the apparatus. The test lasted 5 min after which the chick was killed by decapitation. Various measures of each chick's behaviour were recorded during training and in the choice test. These measures are described in detail elsewhere s and the definitions are summarised for convenience in Table I. Dissection and biochemical procedures After a chick had been killed the brain was quickly removed and the hindbrain discarded. The midbrain was separated from the forebrain and the latter divided into r o o f and base by a diagonal cut extending from the dorsal posterior ridge to the anterior tip. The forebrain roof was further subdivided into anterior and posterior sections (see H o r n et al.19). Each brain region was placed, within 2 rain after decapitation, into individual pots and frozen in solid CO2. The body cavity of each chick was opened and the sex of each chick recorded. The brain samples were kept frozen on solid CO2 or in a deep freeze until they were analysed exactly as described elsewhere 9 by homogenisation, precipitation with trichloroacetic acid (TCA) and radioactive counting. TCA-soluble (pool) and TCA-insoluble (RNA) radioactivity were both expressed as specific radioactivity (disint./min/mg protein). Preliminary experiments have shown that 70-80 ~ of the TCA-insoluble precipitate is found associated with phenol-extracted R N A , whilst most of the rest remains bound to residual protein, possibly associated with the R N A that also remains in this fraction. [14C]Uracil was used in these experiments, rather than [3H]uracil as in our earlier work since use of this carbon-labelled molecule eliminates the problem of exchange of the radioactive label with the hydrogen of water, a rapid process in in vivo experiments using [3H]uracil. Thus, 2 h after the pericardial injection of [3H]uracil,

212 TABLE I! INCORPORATED [i4C]URACIL (ACID-INSOLUBLE) EXPRESSED AS PERCENTAGE OF UNINCORPORATED POOL 1N 4 BRAIN REGIONS FROM 106 CHICKS

Region

Mean

Standard error

Midbrain Base of forebrain Post. roof of forebrain Ant. roof of forebrain

20.57 20.98 23.06 25.18

0.59 0.59 0.87 0.75

63 ~ of the acid-soluble counts in the brain were lost when the acid supernatant was taken to dryness, suggesting they were present as tritiated water. Preliminary experiments using [14C]uracil as precursor showed that after 1 h the acid-insoluble radioactivity was 5.3 ~ of the free pool (unincorporated acid-soluble) radioactivity and by 3 h had increased to 38 ~ of the acid-soluble figure (Haywood, unpublished). The values for the 4 brain regions from the experiment reported in this paper are shown in Table II. In the experiments of this series, the residual pool of unincorporated, acid-soluble radioactivity 120 min after injection was remarkably uniform throughout the brain. Thus, the standardised specific radioactivities of the pool for all 4 brain regions were strongly correlated; even the weakest correlation, between base and posterior roof, is highly significant (Spearman rs = 0.490, P < 0.001). However, the pool specific radioactivity was not correlated with acid-insoluble specific radioactivity in any of the brain regions. The correlation coefficients are shown in Table III and none even approaches statistical significance. It should be noted that the radioactivity in the unincorporated pool is likely to be influenced by events such as changes in blood flow occurring immediately prior to death 12,19. By contrast, radioactivity in the acidinsoluble fraction represents the cumulative effects of events occurring over the 120min period following injection. For these reasons, it is hardly surprising that the correlations shown in Table III are not statistically significant. Nevertheless, sustained depression of the amount of unincorporated precursor over the period of training could greatly reduce the rate of incorporation (see Discussion). Since terminal pool values are by their nature labile, we have not considered correlations between them TABLE lII CORRELATIONS BETWEEN INCORPORATED (ACID-INSOLUBLE) AND UNINCORPORATED (POOL) [14CluRACtL IN EACH OF 4 BRAIN REGIONS FROM 106 CHICKS

Spearman correlation coefficientsare given. Midbrain

Base

Posterior roof

Anterior roof

--0.074

---4).067

--0.082

0.037

When Spearman's rs > 0.192, P < 0.05

213 TABLE IV CORRELATIONS BETWEEN INCORPORATIONS INTO ACID-INSOLUBLE MATERIAL IN

4

BRAIN REGIONS OF

106

CHICKS

Spearman correlation coefficients are given.

Midbrain Base Posterior roof

Base

Posterior roof

Anteriorroof

0.598** ---

0.333**

0.300*

0.164

0.159

--

0.429**

0.01. ** P < 0.001. * P <

and the behavioural measures in the Results section. So far as the specific radioactivities of the acid-insoluble fractions are concerned, Table IV shows the correlations between the values for each of the 4 brain regions. The correlations between incorporation into each region are highly significant with the exception that incorporation into the base is not correlated with incorporation into either of the forebrain regions.

Standardisation procedure and statistical analysis Some of the correlations between raw scores might have been attributable to the substantial batch differences 6 or to differences between the training conditions. For example, the red-trained chicks obtained significantly higher initial approach and preference for familiar scores than the yellow-trained chicks 8. In order to examine correlations which arise from individual differences within batches and within a training condition, it is necessary to eliminate batch differences and training condition differences as sources of variation. This has been done for the behavioural measures in the following way. The means for each measure were calculated for each batch. Then each individual score was expressed as a percentage of the batch mean for that score. In the case of the preference for familiar scores, some of which had negative values, it was necessary to add to each score the distance from the midpoint to one of the ends of the rails, thereby making all scores positive. In order to superimpose the scores of the yellow-trained and red-trained chicks it was necessary to standardise once again. The overall means were calculated for each standardised score from the yellow-trained chicks and the same was done for the redtrained chicks. Finally each score which had already been standardised once to eliminate batch differences was expressed as a percentage of the mean of the standardised scores for that training group. Initially the biochemical data were standardised in the same way as they had been in an earlier publication 10 with the additional feature that the processed scores were then treated in the same way as the behavioural data in order to eliminate training condition as a source of variation. Subsequently, each chick was used as its own reference point (see Discussion) and the specific radioactivities for the two roof regions

214 TABLE V SEQUENCE OF OPERATIONS INVOLVED IN STANDARDISING VALUE

(A) Procedure used for all behavioural a n d biochemical values in order to eliminate differences between batches a n d between training conditions as sources o f variation. 1 2 3 4

O b t a i n raw value for individual. O b t a i n raw batch m e a n for n individuals. Divide raw value by batch mean. Select f r o m all S values those for chicks trained u n d e r condition t (red or yellow light). 5 Obtain m e a n for nt individual used in training condition t. 6 Express St value as percentage of m e a n for training condition.

R1 ~ -- E R / n S~ R1/R St St -- ESt/nt S~ >~ 100/S t

(B) Second procedure used on biochemical values for the two forebrain r o o f regions to obtain values relative to those f r o m the o t h e r brain regions o f the s a m e bird. T h e procedure also eliminated difference between training conditions as a source o f variation. 1 2 3 4 5 6

O b t a i n raw value for i n c o r p o r a t i o n into r o o f region. O b t a i n raw m e a n for m i d b r a i n a n d forebrain base f r o m s a m e bird. Divide raw value by raw m e a n for m i d b r a i n a n d base. Select f r o m all U values those for chicks trained u n d e r condition t. O b t a i n m e a n for nt individuals used in training condition t. Express Ut value as percentage o f m e a n for training condition.

Rt Mt U1 ~ R1/M1 Ut Ut ~- XUt/nt Ut × 100/Ut

were expressed as percentages of the mean specific radioactivity of the midbrain and forebrain base. Differences due to training conditions were then eliminated as before. Worked examples of the standardisation procedures are given in Table V. Spearman correlation coefficients were calculated for all pairs of measurements from 106 chicks used in the experiment. All probability values are 2-tailed unless otherwise stated in the text. RESULTS

No differences between sexes were found in any behavioural or biochemical measure, so data from both sexes were combined in all subsequent analyses. The correlations between 3 behavioural measures and incorporation into acid-insoluble material in the 4 brain regions are shown in Table VI. Training approach latency is positively correlated with incorporation into the midbrain, forebrain base and anterior roof of the forebrain. The longer the chicks took to approach, the higher the specific activity in those regions. Initial approach is negatively correlated with incorporation into the midbrain alone; the more the birds approached in the first 20 min after they had started to approach, the lower the specific radioactivity in this region. Preference for familiar, which is the only direct measure of learning, is positively correlated with incorporation into the anterior roof of the forebrain; the more the birds had learned, the higher was the incorporation in this region alone. However, it should be noted that this association is weak (P < 0.05 for a one-tailed test).

215 TABLE VI CORRELATIONS BETWEEN BEHAVIOUR AND 114C]URACIL INCORPORATION INTO ACID-INSOLUBLE MATERIAL

IN 4 BRAIN REGIONS OF 106 CHICKS Spearman correlation coefficients are given.

Midbrain Approach latency Initial approach Preference for familiar

0.353"* 4.224* --0.150

Base

Posterior roof Anterior roof

0.231 * ---0.088 --0.182

0.091 --0.148 0.093

0.229* 0.021 0.181 §

* P < 0.05. ** P < 0.001. P < 0.05 for one-tailed test.

It seemed possible that the relationship between preference for familiar and incorporation in the anterior forebrain roof might be partially obscured by another factor. Those birds that were quick to respond during training showed a low incorporation into acid-insoluble fractions in the midbrain and forebrain base as well as in the anterior roof of the forebrain (see Table VI). Since the birds which approached most were in general those that learned most s, incorporation into the anterior forebrain roof may have been the result of two opposed effects. One of these effects, shared with other brain regions, may have been to reduce incorporation into the acid-insoluble fraction; the other, specific to the anterior roof may have been to increase incorporation into this fraction. We have eliminated the presumed negative and general effects of high activity during training from the presumed positive and regionally specific effects of learning by expressing the specific radioactivities for the two roof regions as percentages of the specific radioactivities from the forebrain base and midbrain in the TABLE VII CORRELATIONS BETWEEN BEHAVIOURAL MEASURES AND INCORPORATION OF [14C]URACIL INTO ANTERIOR A N D POSTERIOR ROOF REGIONS OF THE FOREBRAIN OF

106

CHICKS

Spearman correlation coefficients are given.

Phase of experiment

Behaviour

Posterior roof

Anterior roof

Training

Approach latency Initial approach Total approach Approach in last session of training

--4). 136 0.006 4.024

--0.062 0.151 0.096

~0.065

4-0.002

Latency Peep calls Movement Preference for familiar

44).082 --0.011 ---0.129 0.178

4--0.092 0.063 --0.010 0.316"

Test

* P < 0.001.

216 L/.I

(3) .H

-~12C

.!

.3100

i 80 e: r4 ~60 "0

e-

Standardized ratios Fig. 2. Preference for familiar scores (means q- standard errors) plotted against incorporation of [14C]uracil into acid-insoluble material in the anterior forebrain roof. The specific radioactivity for the anterior forebrain roof was divided by the mean specific radioactivity of the midbrain and forebrain base for that bird. These standardised ratios, expressed as percentages, were then rank-ordered and divided into 5 groups with 21 chicks in each group except for group 5 which contained 22 chicks. The percentage ranges were as follows: 1 = 51.2-81.8; 2 = 81.9-91.2; 3 = 92.0-102.5; 4 = 102.9112.6; 5 = 112.8-207.0.

same bird (see Materials and Methods). The correlations between the recalculated values for the two roof regions and 8 behavioural measures are shown in Table VII. The only statistically significant correlation coefficient in the table is for the association between preference for familiar and incorporation into the anterior part of the forebrain roof. This correlation is positive and highly significant. In order to present visually the data on which this correlation is based, the values for incorporation into the anterior roof were rank-ordered and divided according to the amount of incorporation into 5 groups. The mean 'preference for familiar' for each of these groups is shown in Fig. 2. DISCUSSION

The experiment described in this paper is part of a series analysing the effects of an imprinting procedure on a number of biochemical measures (see Horn et a l J 9 for review). As briefly summarised in the Introduction of this paper, our earlier studies have suggested that, of all the biochemical changes taking place when a bird is exposed to a flashing light, those occurring rapidly in the anterior part of the forebrain roof are most directly linked with the imprinting process. It must, however, be admitted that the results of each of the control procedures we have devised is open to more than one interpretation. While this is probably inherent in the nature of the problem, the plausibility of any one interpretation of the data is greatly enhanced if the same conclusion

217 is suggested by quite different experimental approaches. In the experiment reported in this paper we have, therefore, attempted to tackle the problem of the specificity of the changes in the forebrain roof in a quite different way from our earlier studies. We have looked for associations between behavioural and biochemical measures of chicks that were trained in identical fashion. When the incorporation rates in the two forebrain regions were standardised with respect to the values in the midbrain and forebrain base of the same bird, a striking result was obtained. Only one correlation between behavioural and biochemical measures is statistically significant and that is the association between the preference for familiar and incorporation of uracil into the acid-insoluble fraction of the anterior forebrain roof. In other words, of all the behavioural measures, only the index of imprinting is positively correlated with a measure of RNA metabolism in the anterior forebrain roof. The association between preference for familiar and incorporation into the posterior forebrain roof is not statistically significant. The biochemical changes in the anterior forebrain roof might reflect the acceleration of, for example, general neuronal development consequent on sensory stimulation. If this had been so we should have expected that the biochemical changes would have been positively correlated with one of the measures of the chicks' behaviour during the training period such as their quickness to respond or their approach activity. However, none of the measures obtained during training is correlated with incorporation into the anterior forebrain roof. Nor, for that matter, is any of the measures of responsiveness, activity or distress in the test correlated with the biochemical measure. The natural variability of the birds has been used, therefore, to dissociate many of the factors which are normally confounded with storage of information during imprinting. The level of statistical significance of the correlation between preference for familiar and incorporation into the anterior forebrain roof does, of course, depend on the method of standardising the biochemical data. When the data were standardised with respect to the batch mean for all the brain samples, the positive correlation between preference for familiar and incorporation into the anterior forebrain roof is much weaker. In addition a quite unexpected result is obtained. The more quickly the chicks started to approach during training, the lower the rates of incorporation into the midbrain, forebiain base and anterior roof of the forebrain. It is likely that an increase in activity associated with vigorous approach movements leads to dilatation of blood vessels in skeletal musclesL As a result, precursor injected into the blood would be distributed over a greater volume of tissue in an active bird than in an inactive one; and the amount of precursor available for incorporation into any one region would be reduced. It also follows that among the trained birds, those that became active shortly after the beginning of training would have less injected precursor available for incorporation into any one region of the body than birds that did not move until much later during training. This interpretation would account for the positive correlations between the training latency and incorporation into the brain. The postulated changes in availability of injected precursor associated with the chicks' approach movements are compatible with evidence obtained by other investigators. A variety of experimental treatments have been shown to influence blood flow in the

218 brain 12 and uptake of precursors into cells in different parts of the body a,')°- 2.),,,4 e~;, 28,29. It seems likely that in our experimental situation activity during training reduces incorporation into the anterior roof, as well as in other regions. However, total approach during training was positively correlated with preference for familiar in the experiment described here (see Bateson and Jaeckel8). Any tendency for increased incorporation in the anterior roof of the forebrain resulting from imprinting would have been offset in the more active birds by a tendency for incorporation to be generally depressed. We consider, therefore, that the measure of incorporation into the anterior forebrain roof represents the outcome of two opposing factors, one specific and one general. By using the incorporation rate in another part of the brain of the same bird as a reference point, the influence of the general factor can be eliminated. It is for this reason, we suggest, that the correlation between the measure of imprinting and the incorporation into the anterior forebrain roof is so highly significant when the specific radioactivity is expressed as a percentage of the mean for the specific radioactivity of the midbrain and base. If the changes in RNA metabolism in the anterior part of the forebrain roof are related to the processes which underlie imprinting, we should expect that interference with RNA and protein synthesis would impair imprinting. Actinomycin D, which blocks RNA synthesis, impairs the effects of training when injected immediately after imprinting 13. Similarly Gervai and Cs~myi14 found that the protein synthesis inhibitor puromycin also impairs imprinting. Gervai and Csfmyi are dubious whether their results can be wholly explained in terms of suppression of protein synthesis since, in independent experiments, they found that the dose of puromycin they used only inhibited protein synthesis by 50 ~. The force ol their objection depends critically on whether puromycin partially inhibits the synthesis of all proteins or wholly suppresses synthesis of half the proteins. In order to block retention only the synthesis of those proteins that are necessary for retention needs to be suppressed. Removal of the forebrain roof would also be expected to impair the effects of imprinting. Negative results from removal prior to imprinting would, however, testify at best to the plasticity of remaining parts of the brain. Unfortunately, no studies have yet been done in which the ablation was performed after training and the chick given a choice between the object with which it bad been trained and a novel object. It is relevant, though, that Benowitz 1~ has shown that ablations of the frontal regions of the chick forebrain impaired retention of an avoidance response but did not affect acquisition in chicks initially trained after surgery. In conclusion, then, the accumulated evidence suggests that the rapidly occurring biochemical changes in the anterior part of the chick's forebrain roof are closely linked with the learning process called imprinting. The various procedures we have used suggest that the biochemical changes are necessary for imprinting. However, we cannot yet be certain that they are exclusively related to it. It is possible, for example, that we have been measuring correlates of some aspect of the chicks' attentiveness to the training objects. The processes involved in selective attention and those exclusively involved in learning have yet to be dissociated.

219 ACKNOWLEDGEMENTS This work was supported by grants f r o m the Science Research C o u n c i l to P P G B a n d G H a n d from the Medical Research C o u n c i l to S P R R . We are grateful to J o h n H a m b l e y , Jeff H a y w o o d , J o a n n a Jaeckel a n d A r u n Sinha for help a n d discussion. We also t h a n k R o b e r t Newhouse for help in the statistical analysis.

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