- Email: [email protected]
Light input and the reversal of functional lateralization in the chicken brain
211
Behavioural Brain Research, 38 (1990) 211-221 Elsevier BBR 01058
Light input and the reversal of functional lateralization in the chicken brain L e s l e y J. R o g e r s Physiology Department, University of New England, Armidale, N.S.W. (Australia) (Received 5 October 1989) (Revised version received 30 January 1990) (Accepted 30 January 1990)
Key words: Brain lateralization; Light input; Reversal; Embryo position; Copulation; Learning; Neural plasticity
During its later stages of development, the chicken embryo is oriented in the egg so that it occludes its left eye with its body and the right eye is positioned so that it can receive light input. This lateralized light input has been shown to play a decisive role in determining the direction of brain lateralization for two behavioural functions, copulation and performance of a visual discrimination task known as the 'pebble floor test', since the direction of lateralization for these functions can be reversed by occluding the right eye of the embryo on day 19/20 of incubation and at the same time exposing the left eye to light. The sensitive period during which this role of lateralized light input influences the lateralization for copulation extends to day 1 posthatching if the eggs are incubated and hatched in darkness, but it is over by day 3. For the pebble floor test the sensitive period is already over by day 1 posthatching. By exposing eggs to light for various times on day 19 of incubation, it was possible to determine that between 2.5 and 6 h of lateralized light exposure is necessary to stabilise the normal direction of lateralization so that it can no longer be reversed by occlusion of the right eye. Thus, in the developing chicken embryo substantial neural reorganization must occur in response to a brief period of lateralized light input.
INTRODUCTION
Many functions of the young chicken brain are lateralized. The right eye is dominant for learning and recall of visual discrimination t a s k s 12,19,3°, while the left eye controls attack and copulation behaviour 26 and visual learning requiring use of topographical cues 3. Summarizing a range of data, Andrew 3 has concluded that the left eye attends to the spatial position of stimuli, while the right eye is more concerned with the details of the stimulus itself. These functional lateralities have also been revealed by unilateral treatment of the hemispheres of the forebrain with cycloheximide, kainic acid or
the excitatory amino acids, glutamate and aspartate 18'23'24. For example, treatment of the left hemisphere with one of these agents on day 2 of posthatching life leads to impaired performance of a visual discrimination learning task requiring search for food grains concealed by a background of pebbles 23, and elevates attack and copulation behaviour 8"18. Similar treatment of the right hemisphere is without effect on these behaviours. This result occurs if the unilateral drug treatment is given at any age between days2 and 5 posthatching 22. The possible mechanisms by which these pharmacological agents act has been considered elsewhere in s o m e detail 8'14'27. It is not known exactly which regions of the forebrain
Correspondence: L.J. Rogers, Physiology Department, University of New England, Armidale, N.S.W. 2351, Australia. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
212 are affected by these agents, but their effects are confined to the telencephalon and, at least, involve an interaction with neurones which process visual information 24,2v. Light experience of the chicken embryo just prior to hatching appears to play an important role in triggering the development of this lateralization of function 21"29. The chicken embryo is oriented in the egg such that, during the later stages of incubation, it occludes its left eye with its body leaving the right eye positioned to receive light input entering the egg through the shell and membranes (Fig. 1). Irrespectively of whether the right eye is open or closed, this eye receives more
light input than the left as the eyelids of the chicken are translucent. Chickens hatched from eggs incubated in darkness during the last few days of embryonic development do not have lateralization for attack, copulation or performance of a visual discrimination task 21. That is, groups of chicks hatched from eggs incubated in darkness were found to display the same levels of attack and copulation behaviour following glutamate treatment of the left hemisphere as those treated with glutamate in the right hemisphere. Nor was there a left-right difference in performance of a visual discrimination task produced by unilateral treatment with glutamate. Strikingly, as little as 2 h of light exposure on day 19 of incubation is sufficient to establish or align the direction of lateralization such that groups of chicks treated with glutamate in the left hemisphere show elevated attack and copulation, whereas those treated in the right hemisphere perform at the low levels characteristic of untreated controls 2~. If the bias in light input received by the embryo's eyes does indeed have a key role in determining the direction of functional lateralization in the chicken brain, rather than having a non-specific effect on brain development, it should be possible to actually reverse the direction of lateralization by occluding the embryo's right eye and allowing light to stimulate the left eye. It is here reported that this reversal can indeed be achieved by monocular input of light to the left eye during the later stages of embryonic development, and also immediately posthatching. In addition, by exposing eggs to light for various periods on day 19 of incubation prior to attempting to reverse the direction of lateralization by right eye occlusion and left eye exposure to light, it has been possible to determine the duration of light exposure necessary to fix, or stabilize, the direction of functional lateralization so that it no longer displays plasticity to lateralized light input. MATERIALS AND METHODS
Fig. 1. Position of the day 19/20embryoin the egg. The shell and membranes have been removed from the air sac end of the egg. Note that the right eye can receive light stimulation when the eye is open or closed because the eyelids are translucent.
Incubation and housing conditions White leghorn crossed with black australorp eggs were incubated for the first 17 days in an
213 automatically turning, forced-draught incubator. They received light exposure every second day when the water level in the incubator was checked. On day 17 the eggs were transferred to small, completely dark, force-draught incubators, and from this time on they were no longer turned. Several batches of eggs contributed to the following treatment groups, an equal number from each batch being distributed to each treatment condition. The first treatment involved manipulating the embryos on day 19/20 of incubation, either just prior to or just after penetration of the beak into the air sac but before pipping. The eggs were removed from the dark incubator and, under very dim lighting conditions, the air sac end of the shell was removed and the head carefully withdrawn from the egg, according to the method of Hamburger and Oppenheim 15. To half of the embryos an eye patch was applied to the right eye and to the other half a patch was applied to the left eye. This latter group served as a control as it mimicked the normal condition of eye occlusion. Before applying the patch the feathers around the eye were dried gently. The patches were made of strongly adhesive black tape, 4 cm × 2 cm, cut to the mid-point half way along the longest side and then twisted into a conical shape. A chin-strap ensured that the patch would not come off. The embryos were then exposed to light (250-350 lux) in an incubator where they remained until some hours after they would normally have hatched. They were placed to lie with their eye patches towards the floor. The patches were removed after 24 h. The survival rate was greater than 95~o. Some chicks, however, required assistance to hatch from the remainder of their egg shells. Between 36 and 40 successfully hatched chicks were assigned to this treatment group and also to each of the treatment groups to follow. A second group of embryos was allowed to remain in the dark incubator until early on the first day posthatching (day 1). At this time, under very dim light, eye patches were applied to their left or right eyes, and they were housed in groups of 3 or 4 with light exposure (250-350 lux). After 24 h the patches were removed.
A third group of chicks hatched from eggs which had received no light exposure, and they were retained in the dark incubator until early on day 3 posthatching. At this age each had an eye patch applied to the left or right eye for 24 h during exposure to light in groups of 3 or 4 in the home cage. In a second series of experiments eggs were incubated as already described, until day 19 when they were divided into 3 groups to be exposed (without manipulation of the embryos) to light for either 1, 2.5 or 6 h. At the end of each of these periods each embryo's head was withdrawn from the egg, under dim lighting conditions, and a patch was applied to the right eye. Light exposure continued with the embryos in an incubator as described above. These patches were removed after 24 h. After hatching the chicks were housed in groups of 3 or 4 for 2 days and then isolated visually from each other. The cages were 22 cm square and 30 cm high with the front wall panel of transparent plastic. Food and water were available ad libitum. Light intensity measurements The amount of light which penetrates the egg shell and membranes was estimated by taking a series of illuminance meter readings (Topcon IM-3) at a range of light intensities both with and without half egg shells plus the membranes of the air sac placed over the photosensitive cell. Glutamate treatment All chicks, except those held in the dark incubator until day 3, were injected on day 2 posthatching. The chicks held in the incubator until day 3 posthatching were treated with glutamate on day 4. The glutamate treatment was used to reveal the presence and direction of lateralization in the chicks following the various conditions of eyepatching. From each incubation condition, the chicks which had received occlusion of the left eye were subdivided into two groups, each containing 8-10 chicks. Those which had received occlusion of the right eye were subdivided similarly. One of these subdivisions received 5 #1 of 100 mM mo-
214 nosodium glutamate injected into the left hemisphere and 5 #1 of physiological saline into the right hemisphere. The other subdivision of chicks received glutamate in the right hemisphere and saline in the left. The drugs were made up in sterile pyrogen-free water, and administered to the middle of each forebrain hemisphere by a technique described previously in detail 29. The chickens were not sexed as earlier studies have shown that the lateralization revealed by unilateral glutamate treatment is present and identical in both males and females 8'21"22. In fact this was the main reason for choosing to reveal the lateralization using the glutamate-administration technique rather than monocular testing. Also chicks perform better when tested binocularly on the behavioural tests. Behavioural tests Copulation. From day 6 to 11 the chicks were tested using a standard hand-thrust test (detailed in Zappia and Rogers 29, as modified from Andrew2). The hand is thrust gently at the chick's chest and then held flat just above floor level with the palm downwards. Juvenile copulation involves mounting the hand, crouching, circling and treading, sometimes with grasping the hand in the beak and pelvic thrusting. Such performance achieves a maximum score of 10 points. The level of copulation is measured according to a rank ordering procedure from 0 to 10. The test is repeated twice, and a mean score calculated. Copulation was scored daily, except for 3 groups which were not scored on day 10. This was the standard means of assessing lateralization in all of the chicks. Attack responses to the moving hand were also scored but they are not presented here as they were the same as for copulation. Pebble floor test. On day 8 or 9 posthatching, some of the groups (see Fig. 3) were also tested on a task requiring search for food crumbles scattered on a background of similarly sized and coloured pebbles stuck down to a plastic floor. This has been described in detail by Rogers et al. 20. Days 8 and 9 posthatching are preferred ages for testing the chicks on this task as by then they are old enough to be well-motivated to feed.
The chicks are deprived of food for 3 h prior to testing. Untreated chicks learn to avoid pebbles and find the food within 60 pecks. Thus, each chick was allowed a total of 60 pecks and only pecks involving new choices of a grain or pebble were scored, not repeated pecks at the same grain or pebble. Error scores (pecks at pebbles) were recorded for each block of 20 pecks. The number of errors in the last block of 20pecks (pecks 41-60) is taken as an indication of learning rate 2°, or performance ability. All the behavioural tests were conducted without the tester knowing previous treatments received by the chicks. The data were analysed by non-parametric statistics as the behavioural scores were derived from a non-ordinal population, as determined previously 29. RESULTS The direction of lateralization for control of copulation was reversed by occlusion of the right eye, coupled with exposure of the left eye to light, for 24 h on either day 19/20 of incubation or day 1 posthatching (Fig. 2, which presents mean scores with standard error bars). The chickens which received this form of manipulation showed elevated levels of copulation when treated with glutamate in the fight hemisphere, but not when treated thus in the left hemisphere. On all days of testing the copulation scores of the chicks treated in the right hemisphere were above those of the chicks treated in the left. The last two days of testing were selected for separate 2-tailed Mann-Whitney U-test comparisons of the group treated with glutamate in the right hemisphere with that treated in the left hemisphere (for day 10, U9.9 = 0, P < 0 . 0 0 2 and, for day 11, U9,9 = 1, P < 0.002). As is clearly evident in Fig. 2, the chicks which received occlusion of the left eye coupled with exposing the right eye to light on either day 19/20 of incubation (i.e. mimicking the natural situation) or on day 1 posthatching displayed the same direction of asymmetry as reported earlier for chicks hatched normally from eggs exposed to light before hatching ~8"2~'29. The copulation
215 RIGHT EYE OCCLUDED
LEFT EYE OCCLUDED A
day E 1 9 / 2 0 8 ¸
6¸
/ 0
5 /
co
/
z
0
Z1
0
i
,
6
7
8
9
10
6
11
7
8
9
10
11
AGE ( D A Y S )
AGE (DAYS) D
day 1 8-
8,
7" 6" 0 O 5o3
5"
Z
_0 i,.-
4
4"
3' 0
2
2,
1
0 6
7
8
9
10
6
11
7
8
day
9
10
11
AGE ( D A Y S )
AGE ( D A Y S ) 3
8
8,
7 6
6,
~5
5
z
4
4-
_J
3~
3-
Q 2"
1.
0 8
7
8
9
AGE (DAYS)
lO
tt
6
7
B
9
10
11
AGE (DAYS)
Fig. 2. The mean copulation scores with standard errors for each group of chicks are presented. The graphs on the left are for the control chicks which had their left eyes occluded by a patch for 24 h. Those on the right are for chicks which had their right eyes patched, the reverse of the normal situation. A and B are the data of chicks which had the patches applied on day 19/20 of incubation; C and D on day 1 posthatching; E and F on day 3 posthatching. Lateralization of this behaviour has been revealed by injecting either the left forebrain hemisphere with glutamate (circles) or the right hemisphere with glutamate (squares). Note the reversed direction of lateralization in the chicks which had their right eyes patched on day 19/20 of incubation or on day 1 posthatching, n = 8-10 per group.
216 scores were elevated following treatment of the left hemisphere with glutamate, but not in those treated in the right hemisphere (for 2-tailed Mann-Whitney U-test comparisons of the left and right glutamate-treated groups on the last two days of testing: after left-eye occlusion on day 19/20, Ug, 9 = 0, P < 0.002, and after left-eye occlusion on day 1, U8,9 = 0.5, P < 0.002). Some of the chicks which were held in the dark incubator until day 3 posthatching and then had the eye patches applied proved to be somewhat more difficult to test for copulation, particularly those treated with glutamate in the fight hemisphere. They showed fear responses to the hand, either attempting to escape or crouching on the floor. Their mean scores plotted in Fig. 2D and E were much lower than those of groups showing elevated copulation in the above experiments (Fig. 2A-D). Furthermore, comparison of the scores of those treated with glutamate in the left hemisphere (on d a y 4 in this case) with those treated in the right revealed no significant difference for either those groups which had received 24 h occlusion of the left eye or of the right eye (for those which had right-eye occlusion, U9,1o = 29.5, P > 0 . 1 0 and left-eye occlusion, Us,9=31.5, P > 0.10). However, examination of the distribution of individual copulation scores in each of these 4 groups indicated bimodal distributions in each case. For example, for the chicks which had received occlusion of the left eye and treatment of glutamate in the left hemisphere, 3 out of 8 chicks had zero mean copulation scores and the remaining 5 had scores above 5.0. These scores were consistent across testing days for each individual. Similarly, of those which had had left eye occlusion and treatment of the right hemisphere, 4 out of 9 had mean scores below 2.0 and the remaining 5 had scores above 5.0. The same pattern was evident in the chicks which had received occlusion of their right eyes. The numbers in each group are too low to apply statistical tests for bimodality, and it would not be enlightening to lump the data for all of these groups, but it should be noted that the bimodal pattern has been reported earlier for chicks hatched from eggs incubated in darkness and then brought into the light without eye patching on day 1 posthatching 29.
The direction of lateralization for perfbrmance in the pebble floor test is also reversed by occlusion of the right eye for 24 h on day 19/20 of incubation (Fig. 3B). After this manipulation, glutamate treatment of the right hemisphere impairs performance while treatment of the left does not; the right-treated group made significantly more pecks at pebbles in the last 20 pecks (Us,~ = 8, 0,002 < P < 0.02, 2-tailed Mann-Whitney Utest). Controls, which received occlusion of the left eye on day 19/20, showed the usual direction of lateralization for this task found in chicks hatched from unmanipulated eggs exposed to light; viz., impaired performance following treatment of the left hemisphere with glutamate and no effect of treating the right (U~,8 = 9.5, P = 0.009, 1-tailed Mann-Whitney U-test for the difference in the number of pecks at pebbles in the last 20 pecks, Fig. 3A). The number of pecks at pebbles in the last 20 pecks was analysed, but it can also be seen in Fig. 3 A - C that the slopes of the curves differ in a manner consistent with these scores. Unlike the data for copulation, however, occlusion of the right eye on day 1 posthatching failed to reverse the direction of lateralization for visual discrimination performance (Fig. 3C). The chicks treated in the left hemisphere showed impaired performance (Usa o = 0, P < 0.002, 2tailed Mann-Whitney U-test for the difference in the number of pecks at pebbles in the last 20 pecks). For those chicks held in the dark incubator until day 3 posthatching and then given right eye occlusion followed by glutamate treatment, there was also no reversal of the direction of lateralization. Those treated in the left hemisphere avoided pecking pebbles significantly more slowly than those treated in the right (Ug,m = 16.5, 0.02 < P < 0.05, 2-tailed Mann-Whitney U-test for the difference in the number of pecks at pebbles in the last 20 pecks; Fig. 3D). However, in this case the group treated in the left hemisphere did show some avoidance of pebbles over the testing period. It should be noted that there were no differences between any of these groups in the time taken to complete the 60 pecks.
217 A 18.
18"
LEFT EYE OCCLUDED day
RIGHT EYE OCCLUDED day E 19/20
E 19/20
16 ¸
16
14
14
12-
12 10-
O~ ,'r 1 0 . 0 EE rt" 8uJ
\
8-
\ \
6-
\
4. 2' 0 1
2
i 1
3
2
3
BLOCKS OF 20 PECKS
BLOCKS OF 20 PECKS D 18-
RIGHT EYE OCCLUDED day 1
18-
16-
16-
14 °
14.
12"
12.
RIGHT EYE OCCLUDED day 3
10-
rr
W
x\
\
0 8"
8
6'
6
\x
42-
2
0
0 1
2
3
BLOCKS OF 20 PECKS
i
I
2
3
BLOCKS OF 20 PECKS
Fig. 3. Performance on the pebble floor is presented as the mean number of errors (pecks at pebbles) in each block of 20 pecks with standard error scores. A fall in the curve indicates increasing avoidance of pebbles. The symbols are as in Fig. 2; glutamate in the left (circles), glutamate in the right (squares). Note the reversal of lateralization in those chicks which had had the right eye patched on day 19/20 of incubation (B, compared to A), but not in the groups which received this manipulation on days 1 or 3 posthatching (C and D). n = 8-10 per group.
The final procedure was aimed at finding out the number of hours of monocular light exposure, given to unmanipulated embryos, necessary to stabilise the direction oflateralization and prevent its subsequent reversal by occlusion of the right eye for 24 h on day 19/20. As shown in Fig. 4, occlusion of the right eye reversed the direction of lateralization in those embryos which had been exposed to 1 and 2.5 h of light while still in their normal position in the egg; copulation scores were elevated following treatment of the right hemisphere but not the left (Fig. 4A and B; comparison of the left and right scores using 2-tailed
Mann-Whitney U-tests gave, after 1 h of light, U8.9=6.5, 0 . 0 0 2 < P < 0 . 0 2 for d a y l 0 and U8,9 = 4.5, P < 0.002 for day 11, and after 2.5 h of light, Us,8 = 2, P = 0.000 for both days 10 and 11). In contrast, occlusion of the right eye failed to reverse the normal direction of lateralization in the group of eggs exposed to light 6 h prior to the monocular exposure. In these groups, treatment of the left hemisphere with glutamate elevated copulation, but treatment of the right did not (U8.8 = 6.5, P = 0.05 for left-right 2-tailed comparisons on days 10 and 11). The amount of light which penetrates the egg
218 t~
6~
6-
5"
0
/"
4-
F- 4 '~ i
/
//
3-
3 -
2-
6
7
8
9
10
11
6
7
AGE (DAYS)
8
9
AGE (DAYS)
10
11
6
r
e
9
'~
~1
AGE (DAYS}
Fig. 4. This figure presents data for attempted reversal of lateralization by occluding the right eye on day 19/20 of incubation following exposure of the eggs to light for 1, 2.5 or 6 h immediately prior to this manipulation. The symbols and scores are as in Fig. 2. Note the reversed direction of lateralization (opposite to that normally present) in A and B, but not C.
shell and membranes was measured at various levels of ambient illumination, and it was consistently found that only between 6% (for brown eggs) and 11% (for white eggs) of the light falling on the surface of the egg penetrates the shell and membranes. Thus, in this last experiment the eggs were exposed to between 250 and 350 lux of light and the embryos inside received in the region of 25-35 lux. DISCUSSION
The direction of functional lateralization can, as these experiments show, be reversed by occluding the right eye for 24 h while allowing the left eye to receive light stimulation. Occlusion of the right eye on day 19/20 of embryonic life has been shown to reverse the direction of lateralization for control of both copulation and performance in the pebble floor test: in these animals glutamate treatment of the right, but not the left, hemisphere elevates copulation and impairs performance in the pebble floor test. Controls, which received the same operative procedure but with occlusion of the left eye instead of the right, and so mimicked the normal eye occlusion which occurs during the later phases of development in chick embryo, exhibited the same direction of lateralization for these behaviours as found normally (i.e. that present in the brains of groups of chicks hatched from eggs which have received at
least 2 h of light stimulation during the last phases of embryonic development). They showed elevated copulation and impaired performance in the pebble floor test following glutamate treatment of the left, but not the fight, hemisphere. These results, I believe, rather conclusively confirm my original hypotheses 23 that functional lateralization in the chicken brain, for these behaviours at least, results from lateralized input of light to the eyes during the developmental period when visual connections to the forebrain are being established. Although the first connections between retinal ganglion cell axons and cells in the optic tecta begin to form as early as day 6 of embryonic life, these connections continue to develop by arborization and retraction of their axonal end terminals until around embryonic day 16 or 17, and the chick visual system is not considered to be in any way functionally mature until day 18 of embryonic development28. On day 18 of incubation an electroretinogram of embryonic form can be detected and the first behavioural responses to light stimulation can be detected. On day 19 of incubation the electroretinogram assumes its posthatching form and light-stimulated potentials can be detected for the first time in the contralateral forebrain hemisphere 11. One would therefore expect the critical period for the influence of lateralized light input on the subsequent lateralization of brain function to
219 have its onset on day 18 or 19 of incubation, although this has not been investigated experimentally. The experiments presented here were designed to determine the end point of a sensitive period for the role of lateralized light stimulation. For lateralization of performance of the pebble floor test this sensitive period ended by the time of hatching. Right eye occlusion on day 1 posthatching, and also on day 3 posthatching, is ineffective in reversing the lateralization for this behaviour in chicks incubated and hatched in darkness. In contrast, the sensitive period during which lateralized light input can influence subsequent lateralization of brain function concerned with copulation behaviour extends into posthatching life. Right eye occlusion coupled with light exposure of the left eye was found to reverse the direction of lateralization for copulation when applied on day 1 posthatching to chicks hatched in darkness from eggs incubated in darkness. Similar right eye occlusion on day 3 posthatching failed to reverse the direction of lateralization for copulation. In this case there were no overall group differences between those injected in the left and those in the right hemispheres irrespective of whether the chicks had received occlusion of their left or right eyes on day 3. Nevertheless, apparent bimodality of the individual scores in each of these groups may be taken to indicate that each individual retained lateralization for copulation although there was no common alignment amongst the members of a given :~oup (i.e. no group bias). In other words, these ,:hicks gave the same results as reported previously by Zappia and Rogers 29 for chicks ihatched from eggs incubated in darkness and brought into the light after hatching so that at no ;ime did they receive monocular light input. Thus, it would appear that by day 3 posthatching monocular, lateralized light input, be it to the left or right eye, has no effect, and the chickens behave merely as if they had not received it. The sensitive period for lateralized light input to determine the direction of lateralization for copulation has ended by day 3 posthatching, and chicks kept in darkness until this age retain lateralization, but its direction is randomly distributed amongst individuals. Therefore, in the normally developing
embryo exposed to light prior to hatching, the lateralized stimulation does not establish the presence of lateralization for copulation but, rather, aligns it in a particular direction so that all (or most) individuals in the group have a common direction of bias. The lateralization of brain function involved in performance of the pebble floor test appears to differ from this. Lateralized light input certainly plays a role in determining the direction of this form oflateralization, since right eye occlusion on day 19/20 can reverse its normal direction. Yet, rather than showing an absence of lateralization at group level, the group of chicks which received right eye occlusion on day 1, and less clearly those which received it on day 3, displayed the normal direction of lateralization. All individuals were lateralized, but in the normal direction and thus apparently uninfluenced by the right eye occlusion. One might therefore suggest that lateralization for visual discrimination learning does not require asymmetrical light input to ensure its common alignment amongst individuals in the group, but nevertheless, during the sensitive period at the end of the incubation period, its direction can be reversed by monocular, lateralized light input. This difference between the effect of light stimulation on lateralization of performance of the pebble floor test and copulation behaviour may relate to the different neural pathways involved in each, and the relative dependence of their development on genetic and environmental factors. Indeed, it has already been shown that glutamate treatment impairs performance of the pebble floor test only when its time of action is coupled with the chick viewing certain visual stimuli 27, whereas glutamate's effect in elevating copulation does not have this visual requirement 24. Copulation behaviour is well known to involve hypothalamic circuits 5 probably influenced by the forebrain 8; whereas visual discrimination performance must involve processing in either one or both of the visual systems of the forebrain (i.e. the tectorotundal-ectostriatal system and the thalamohyperstriatal system). In fact, a structural asymmetry is known to exist in the thalamo-hyperstriatal visual system of young chicks 7,25. The projections from the left side of the thalamus (fed by the
220 right eye only) to the hyperstriatum are more numerous than those from the right side of the thalamus (fed by the left eye only). This structural asymmetry is also dependent on lateralized light input as it can be reversed by occluding the right eye on day 19/20 while exposing the left to light 25. Asymmetry in these visual projections may underly the lateralization for visual discrimination performance, but it cannot be the complete explanation because the structural asymmetry is present in young males and not females 1, whereas glutamate treatment reveals functional asymmetry in both sexes. It would seem that monocular light stimulation during this sensitive period of development stimulates growth of neural projections fed either directly or indirectly by the stimulated eye. One might argue that in normal situations light received by the right eye stimulates developmental processes to occur in the left hemisphere in advance of those in the right, leading to their susceptibility to glutamate treatment later on 22. However, this may well be an oversimplified view, since each eye is not simply connected to the contralateral side of the brain, crossing over occurring in the tectal commissure and the supraoptic decussation 9. Lastly, given that as little as 2 h of light stimulation is sufficient to determine the direction of lateralization for copulation in the normal situation 21, it was considered of interest to determine the duration of light exposure necessary to prevent subsequent reversal of the lateralization by right eye occlusion. After 1 or 2.5 h of prior exposure of the eggs to light (250-350 lux at the egg's surface, but only 25-35 lux inside the egg), it was still possible to reverse the lateralization for copulation by occluding the right eye for 24 h on day 19/20 of incubation, but this could not be done after 6 h of prior exposure. That is, it takes between 2.5 and 6 h of light to stabilize the (normal) direction of lateralization so that it can no longer be reversed by this manipulation. Hence, while the sensitive period for the role of light in determining lateralization of copulation extends beyond day 1 posthatching when the eggs and hatchlings are held in darkness, monocular light exposure prior to hatching closes that sensitive period. This has similarities to the sensitive period
for imprinting, which remains open for 1 week if the chicks are dark-reared, but is closed earlier by exposure to the imprinting stimulus 17 It should be noted that it takes a shorter period of exposure to lateralized light to align the direction of lateralization (no more than 2 h) than it does to stabilise, or fix, it so that it can no longer be reversed by the converse light exposure (between 2.5 and 6 h). This makes biological sense. These experiments have demonstrated a role for non-patterned, white light on the organisation of lateralization in the forebrain, and it is perhaps surprising that an intensity as low as 25-35 lux is sufficient to bring about these effects. At least, this appears to be the intensity of light which, in these experiments, reached the embryo's right eye through the egg shell. These appear to be the first examples of rather dramatic reorganization of brain functions based on such a brief environmental exposure. Falling within a sensitive period, they have much in common with the previously demonstrated effects of visual stimulation on organization of the visual cortex in kittens 6. It should be noted that Jones and Horn 13 have reported that exposure of chicks for as little as 3 h to a flickering light on day 1 posthatching leads to increased effectiveness in synaptic transmission in the hyperstriatum, but not the ectostriatum, as measured by evoked potentials. Other experiments which have looked at the effects of monocularity in birds have involved weeks or months of monocular eye occlusion 4"16, although admittedly these studies have looked for structural or biochemical changes in the brain following monocularity rather than functional effects. Functional changes may well be more light-sensitive, but it should be remembered that they are coupled with structural reorganization of the thalamo-hyperstriatal visual projections of the brain.
ACKNOWLEDGEMENT
This research was funded by an Australian Research Council Grant A18830646.
221 REFERENCES 1 Adret, P. and Rogers, L.J., Sex difference in the visual projections of young chicks: a quantitative study of the thalamofugal pathway, Brain Res., 478 (1989) 59-73. 2 Andrew, R.J., Precocious adult behaviour in the young chick, Anita. Behav., 14 (1966) 485-500. 3 Andrew, R.J., The development of visual lateralization in the domestic chick, Behav. Brain. Res., 29 (1988) 201-209. 4 Bagnoli, P., Barsellotti, R., Pellegrini, M. and Alesc, R., Norepinephrine levels in developing pigeon brain: effect of monocular deprivation in the Wulst noradrenergic system, Dev. Brain. Res., 10 (1983) 243-250. 5 Barfield, R.J., Induction of copulatory behavior by intracranial placement of androgen in capons, Am. Zool., 4 (1964) 301. 6 Blakemore, C. and Cooper, G.F., Development of the brain depends on the visual environment, Nature (Lond.), 228 (1970) 477-478. 7 Boxer, M.I. and Stanford, D., Projections to the posterior visual hyperstriatal region of the chick: an HRP study, Exp. Brain Res., 57 (1985) 494-498. 8 Bullock, S.P. and Rogers, L.J., Glutamate-induced asymmetry in the sexual and aggressive behaviour of young chickens, Pharm. Biochem. Behav., 24 (1986) 549-554. 9 Curnod, M., Commissural pathways in interhemispheric transfer of visual information in the pigeon. In F.O. Schmitt and F.G. Worden (Eds.), The Neurosciences, Third Study Program, MIT Press, Cambridge, MA, 1973, pp. 21-29. 10 Ehrlich, D. and Saleh, C.N., The effect of eye enucleation on the number of fibres in the supraoptic decussation of posthatch chicks (Gallusgallus), Neurosci. Lett., 60 (1985) 349-355. 11 Freeman, B.M. and Vince, M.A., Development of the Avian Embryo, Chapman and Hall, London, 1974. 12 Gaston, K.E. and Gaston, M.G., Unilateral memory after binocular discrimination training: left hemisphere dominance in the chick? Brain Res., 303 (1984) 170-193. 13 Jones, S.J. and Horn, G., Effects of visual experience on photically evoked potentials recorded in the chick forebrain, Brain Res., 159 (1978) 297-306. 14 Hambley, J.W. and Rogers, L.J., Retarded learning induced by intracerebral administration of amino acids in the neonatal chick, Neuroscience, 4 (1979) 677-684. 15 Hamburger, V. and Oppenheim, R., Prehatching motility
and hatching behaviour in the chick, J. Exp. Zool., 166 (1967) 171-204. 16 Herrmann, K. and Bischoff, H.J.,Monocular deprivation affects neurone size in the ectostriatum of the zebra finch brain, Brain Res., 379 (1986) 143-146. 17 Horn, G., Memory., Imprinting and the Brain: An Inquiry into Mechanisms, Clarendon, Oxford, 1985. 18 Howard, K.J., Rogers, L.J. and Boura, A.L.A., Functional lateralization of the chick forebrain revealed by use of intracranial glutamate, Brain Res., 188 (1980) 369-382. 19 Mench, J.A. and Andrew, R.J., Lateralization of a food search task in the domestic chick, Behav. Neural Biol., 46 (1986) 107-114. 20 Rogers, L.J., Drennen, H.D. and Mark, R.F., Inhibition of memory formation in the imprinting period: irreversible action ofcycloheximide in young chickens, Brain Res., 79 (1974) 213-233. 21 Rogers, L.J., Light experience and asymmetry of brain function in chickens, Nature (Lond), 297 (1982) 223-225. 22 Rogers, L.J., Lateralization of learning in chicks, Adv. Study Behav., 16 (1986) 147-189. 23 Rogers, L.J. and Anson, J.M., Lateralization of function in the chicken forebrain, Pharm. Biochem. Behav., 10 (1979) 679-686. 24 Rogers, L.J. and Hambley, J.W., Specific and non-specific effects of neuro-excitatory amino acids on learning and other behaviours in the chicken, Behav. Brain Res., 4 (1982) 1-18. 25 Rogers, L.J. and Sink, H.S., Transient asymmetry in the projections of the rostral thalamus to the visual hyperstriatum of the chicken, and reversal of its direction by light exposure, Exp. Brain Res., 70 (1988) 378-384. 26 Rogers, L.J., Zappia, J.V. and Bullock, S.P., Testosterone and eye-brain asymmetry for copulation in chickens, Experientia, 41 (1985) 1447-1449. 27 Sdraulig, R., Rogers, L.J. and Boura, A.L.A., Glutamate and specific perceptual input interact to cause retarded learning in chicks, Physiol. Behav., 24 (1980) 493-500. 28 Thanos, S. and Bonhoeffer, F., Axonal arborization in the developing chick retinotectal system, J. Camp. Neurol., 261 (1987) 155-164. 29 Zappia, J.V. and Rogers, L.J., Light experience during development affects asymmetry of forebrain function in chickens, Dev. Brain Res., 11 (1983) 93-106. 30 Zappia, J.V. and Rogers, L.J., Sex differences and reversal of brain asymmetry by testosterone in chickens, Behav. Brain Res., 23 (1987) 261-267.
Behavioural Brain Research, 38 (1990) 211-221 Elsevier BBR 01058
Light input and the reversal of functional lateralization in the chicken brain L e s l e y J. R o g e r s Physiology Department, University of New England, Armidale, N.S.W. (Australia) (Received 5 October 1989) (Revised version received 30 January 1990) (Accepted 30 January 1990)
Key words: Brain lateralization; Light input; Reversal; Embryo position; Copulation; Learning; Neural plasticity
During its later stages of development, the chicken embryo is oriented in the egg so that it occludes its left eye with its body and the right eye is positioned so that it can receive light input. This lateralized light input has been shown to play a decisive role in determining the direction of brain lateralization for two behavioural functions, copulation and performance of a visual discrimination task known as the 'pebble floor test', since the direction of lateralization for these functions can be reversed by occluding the right eye of the embryo on day 19/20 of incubation and at the same time exposing the left eye to light. The sensitive period during which this role of lateralized light input influences the lateralization for copulation extends to day 1 posthatching if the eggs are incubated and hatched in darkness, but it is over by day 3. For the pebble floor test the sensitive period is already over by day 1 posthatching. By exposing eggs to light for various times on day 19 of incubation, it was possible to determine that between 2.5 and 6 h of lateralized light exposure is necessary to stabilise the normal direction of lateralization so that it can no longer be reversed by occlusion of the right eye. Thus, in the developing chicken embryo substantial neural reorganization must occur in response to a brief period of lateralized light input.
INTRODUCTION
Many functions of the young chicken brain are lateralized. The right eye is dominant for learning and recall of visual discrimination t a s k s 12,19,3°, while the left eye controls attack and copulation behaviour 26 and visual learning requiring use of topographical cues 3. Summarizing a range of data, Andrew 3 has concluded that the left eye attends to the spatial position of stimuli, while the right eye is more concerned with the details of the stimulus itself. These functional lateralities have also been revealed by unilateral treatment of the hemispheres of the forebrain with cycloheximide, kainic acid or
the excitatory amino acids, glutamate and aspartate 18'23'24. For example, treatment of the left hemisphere with one of these agents on day 2 of posthatching life leads to impaired performance of a visual discrimination learning task requiring search for food grains concealed by a background of pebbles 23, and elevates attack and copulation behaviour 8"18. Similar treatment of the right hemisphere is without effect on these behaviours. This result occurs if the unilateral drug treatment is given at any age between days2 and 5 posthatching 22. The possible mechanisms by which these pharmacological agents act has been considered elsewhere in s o m e detail 8'14'27. It is not known exactly which regions of the forebrain
Correspondence: L.J. Rogers, Physiology Department, University of New England, Armidale, N.S.W. 2351, Australia. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
212 are affected by these agents, but their effects are confined to the telencephalon and, at least, involve an interaction with neurones which process visual information 24,2v. Light experience of the chicken embryo just prior to hatching appears to play an important role in triggering the development of this lateralization of function 21"29. The chicken embryo is oriented in the egg such that, during the later stages of incubation, it occludes its left eye with its body leaving the right eye positioned to receive light input entering the egg through the shell and membranes (Fig. 1). Irrespectively of whether the right eye is open or closed, this eye receives more
light input than the left as the eyelids of the chicken are translucent. Chickens hatched from eggs incubated in darkness during the last few days of embryonic development do not have lateralization for attack, copulation or performance of a visual discrimination task 21. That is, groups of chicks hatched from eggs incubated in darkness were found to display the same levels of attack and copulation behaviour following glutamate treatment of the left hemisphere as those treated with glutamate in the right hemisphere. Nor was there a left-right difference in performance of a visual discrimination task produced by unilateral treatment with glutamate. Strikingly, as little as 2 h of light exposure on day 19 of incubation is sufficient to establish or align the direction of lateralization such that groups of chicks treated with glutamate in the left hemisphere show elevated attack and copulation, whereas those treated in the right hemisphere perform at the low levels characteristic of untreated controls 2~. If the bias in light input received by the embryo's eyes does indeed have a key role in determining the direction of functional lateralization in the chicken brain, rather than having a non-specific effect on brain development, it should be possible to actually reverse the direction of lateralization by occluding the embryo's right eye and allowing light to stimulate the left eye. It is here reported that this reversal can indeed be achieved by monocular input of light to the left eye during the later stages of embryonic development, and also immediately posthatching. In addition, by exposing eggs to light for various periods on day 19 of incubation prior to attempting to reverse the direction of lateralization by right eye occlusion and left eye exposure to light, it has been possible to determine the duration of light exposure necessary to fix, or stabilize, the direction of functional lateralization so that it no longer displays plasticity to lateralized light input. MATERIALS AND METHODS
Fig. 1. Position of the day 19/20embryoin the egg. The shell and membranes have been removed from the air sac end of the egg. Note that the right eye can receive light stimulation when the eye is open or closed because the eyelids are translucent.
Incubation and housing conditions White leghorn crossed with black australorp eggs were incubated for the first 17 days in an
213 automatically turning, forced-draught incubator. They received light exposure every second day when the water level in the incubator was checked. On day 17 the eggs were transferred to small, completely dark, force-draught incubators, and from this time on they were no longer turned. Several batches of eggs contributed to the following treatment groups, an equal number from each batch being distributed to each treatment condition. The first treatment involved manipulating the embryos on day 19/20 of incubation, either just prior to or just after penetration of the beak into the air sac but before pipping. The eggs were removed from the dark incubator and, under very dim lighting conditions, the air sac end of the shell was removed and the head carefully withdrawn from the egg, according to the method of Hamburger and Oppenheim 15. To half of the embryos an eye patch was applied to the right eye and to the other half a patch was applied to the left eye. This latter group served as a control as it mimicked the normal condition of eye occlusion. Before applying the patch the feathers around the eye were dried gently. The patches were made of strongly adhesive black tape, 4 cm × 2 cm, cut to the mid-point half way along the longest side and then twisted into a conical shape. A chin-strap ensured that the patch would not come off. The embryos were then exposed to light (250-350 lux) in an incubator where they remained until some hours after they would normally have hatched. They were placed to lie with their eye patches towards the floor. The patches were removed after 24 h. The survival rate was greater than 95~o. Some chicks, however, required assistance to hatch from the remainder of their egg shells. Between 36 and 40 successfully hatched chicks were assigned to this treatment group and also to each of the treatment groups to follow. A second group of embryos was allowed to remain in the dark incubator until early on the first day posthatching (day 1). At this time, under very dim light, eye patches were applied to their left or right eyes, and they were housed in groups of 3 or 4 with light exposure (250-350 lux). After 24 h the patches were removed.
A third group of chicks hatched from eggs which had received no light exposure, and they were retained in the dark incubator until early on day 3 posthatching. At this age each had an eye patch applied to the left or right eye for 24 h during exposure to light in groups of 3 or 4 in the home cage. In a second series of experiments eggs were incubated as already described, until day 19 when they were divided into 3 groups to be exposed (without manipulation of the embryos) to light for either 1, 2.5 or 6 h. At the end of each of these periods each embryo's head was withdrawn from the egg, under dim lighting conditions, and a patch was applied to the right eye. Light exposure continued with the embryos in an incubator as described above. These patches were removed after 24 h. After hatching the chicks were housed in groups of 3 or 4 for 2 days and then isolated visually from each other. The cages were 22 cm square and 30 cm high with the front wall panel of transparent plastic. Food and water were available ad libitum. Light intensity measurements The amount of light which penetrates the egg shell and membranes was estimated by taking a series of illuminance meter readings (Topcon IM-3) at a range of light intensities both with and without half egg shells plus the membranes of the air sac placed over the photosensitive cell. Glutamate treatment All chicks, except those held in the dark incubator until day 3, were injected on day 2 posthatching. The chicks held in the incubator until day 3 posthatching were treated with glutamate on day 4. The glutamate treatment was used to reveal the presence and direction of lateralization in the chicks following the various conditions of eyepatching. From each incubation condition, the chicks which had received occlusion of the left eye were subdivided into two groups, each containing 8-10 chicks. Those which had received occlusion of the right eye were subdivided similarly. One of these subdivisions received 5 #1 of 100 mM mo-
214 nosodium glutamate injected into the left hemisphere and 5 #1 of physiological saline into the right hemisphere. The other subdivision of chicks received glutamate in the right hemisphere and saline in the left. The drugs were made up in sterile pyrogen-free water, and administered to the middle of each forebrain hemisphere by a technique described previously in detail 29. The chickens were not sexed as earlier studies have shown that the lateralization revealed by unilateral glutamate treatment is present and identical in both males and females 8'21"22. In fact this was the main reason for choosing to reveal the lateralization using the glutamate-administration technique rather than monocular testing. Also chicks perform better when tested binocularly on the behavioural tests. Behavioural tests Copulation. From day 6 to 11 the chicks were tested using a standard hand-thrust test (detailed in Zappia and Rogers 29, as modified from Andrew2). The hand is thrust gently at the chick's chest and then held flat just above floor level with the palm downwards. Juvenile copulation involves mounting the hand, crouching, circling and treading, sometimes with grasping the hand in the beak and pelvic thrusting. Such performance achieves a maximum score of 10 points. The level of copulation is measured according to a rank ordering procedure from 0 to 10. The test is repeated twice, and a mean score calculated. Copulation was scored daily, except for 3 groups which were not scored on day 10. This was the standard means of assessing lateralization in all of the chicks. Attack responses to the moving hand were also scored but they are not presented here as they were the same as for copulation. Pebble floor test. On day 8 or 9 posthatching, some of the groups (see Fig. 3) were also tested on a task requiring search for food crumbles scattered on a background of similarly sized and coloured pebbles stuck down to a plastic floor. This has been described in detail by Rogers et al. 20. Days 8 and 9 posthatching are preferred ages for testing the chicks on this task as by then they are old enough to be well-motivated to feed.
The chicks are deprived of food for 3 h prior to testing. Untreated chicks learn to avoid pebbles and find the food within 60 pecks. Thus, each chick was allowed a total of 60 pecks and only pecks involving new choices of a grain or pebble were scored, not repeated pecks at the same grain or pebble. Error scores (pecks at pebbles) were recorded for each block of 20 pecks. The number of errors in the last block of 20pecks (pecks 41-60) is taken as an indication of learning rate 2°, or performance ability. All the behavioural tests were conducted without the tester knowing previous treatments received by the chicks. The data were analysed by non-parametric statistics as the behavioural scores were derived from a non-ordinal population, as determined previously 29. RESULTS The direction of lateralization for control of copulation was reversed by occlusion of the right eye, coupled with exposure of the left eye to light, for 24 h on either day 19/20 of incubation or day 1 posthatching (Fig. 2, which presents mean scores with standard error bars). The chickens which received this form of manipulation showed elevated levels of copulation when treated with glutamate in the fight hemisphere, but not when treated thus in the left hemisphere. On all days of testing the copulation scores of the chicks treated in the right hemisphere were above those of the chicks treated in the left. The last two days of testing were selected for separate 2-tailed Mann-Whitney U-test comparisons of the group treated with glutamate in the right hemisphere with that treated in the left hemisphere (for day 10, U9.9 = 0, P < 0 . 0 0 2 and, for day 11, U9,9 = 1, P < 0.002). As is clearly evident in Fig. 2, the chicks which received occlusion of the left eye coupled with exposing the right eye to light on either day 19/20 of incubation (i.e. mimicking the natural situation) or on day 1 posthatching displayed the same direction of asymmetry as reported earlier for chicks hatched normally from eggs exposed to light before hatching ~8"2~'29. The copulation
215 RIGHT EYE OCCLUDED
LEFT EYE OCCLUDED A
day E 1 9 / 2 0 8 ¸
6¸
/ 0
5 /
co
/
z
0
Z1
0
i
,
6
7
8
9
10
6
11
7
8
9
10
11
AGE ( D A Y S )
AGE (DAYS) D
day 1 8-
8,
7" 6" 0 O 5o3
5"
Z
_0 i,.-
4
4"
3' 0
2
2,
1
0 6
7
8
9
10
6
11
7
8
day
9
10
11
AGE ( D A Y S )
AGE ( D A Y S ) 3
8
8,
7 6
6,
~5
5
z
4
4-
_J
3~
3-
Q 2"
1.
0 8
7
8
9
AGE (DAYS)
lO
tt
6
7
B
9
10
11
AGE (DAYS)
Fig. 2. The mean copulation scores with standard errors for each group of chicks are presented. The graphs on the left are for the control chicks which had their left eyes occluded by a patch for 24 h. Those on the right are for chicks which had their right eyes patched, the reverse of the normal situation. A and B are the data of chicks which had the patches applied on day 19/20 of incubation; C and D on day 1 posthatching; E and F on day 3 posthatching. Lateralization of this behaviour has been revealed by injecting either the left forebrain hemisphere with glutamate (circles) or the right hemisphere with glutamate (squares). Note the reversed direction of lateralization in the chicks which had their right eyes patched on day 19/20 of incubation or on day 1 posthatching, n = 8-10 per group.
216 scores were elevated following treatment of the left hemisphere with glutamate, but not in those treated in the right hemisphere (for 2-tailed Mann-Whitney U-test comparisons of the left and right glutamate-treated groups on the last two days of testing: after left-eye occlusion on day 19/20, Ug, 9 = 0, P < 0.002, and after left-eye occlusion on day 1, U8,9 = 0.5, P < 0.002). Some of the chicks which were held in the dark incubator until day 3 posthatching and then had the eye patches applied proved to be somewhat more difficult to test for copulation, particularly those treated with glutamate in the fight hemisphere. They showed fear responses to the hand, either attempting to escape or crouching on the floor. Their mean scores plotted in Fig. 2D and E were much lower than those of groups showing elevated copulation in the above experiments (Fig. 2A-D). Furthermore, comparison of the scores of those treated with glutamate in the left hemisphere (on d a y 4 in this case) with those treated in the right revealed no significant difference for either those groups which had received 24 h occlusion of the left eye or of the right eye (for those which had right-eye occlusion, U9,1o = 29.5, P > 0 . 1 0 and left-eye occlusion, Us,9=31.5, P > 0.10). However, examination of the distribution of individual copulation scores in each of these 4 groups indicated bimodal distributions in each case. For example, for the chicks which had received occlusion of the left eye and treatment of glutamate in the left hemisphere, 3 out of 8 chicks had zero mean copulation scores and the remaining 5 had scores above 5.0. These scores were consistent across testing days for each individual. Similarly, of those which had had left eye occlusion and treatment of the right hemisphere, 4 out of 9 had mean scores below 2.0 and the remaining 5 had scores above 5.0. The same pattern was evident in the chicks which had received occlusion of their right eyes. The numbers in each group are too low to apply statistical tests for bimodality, and it would not be enlightening to lump the data for all of these groups, but it should be noted that the bimodal pattern has been reported earlier for chicks hatched from eggs incubated in darkness and then brought into the light without eye patching on day 1 posthatching 29.
The direction of lateralization for perfbrmance in the pebble floor test is also reversed by occlusion of the right eye for 24 h on day 19/20 of incubation (Fig. 3B). After this manipulation, glutamate treatment of the right hemisphere impairs performance while treatment of the left does not; the right-treated group made significantly more pecks at pebbles in the last 20 pecks (Us,~ = 8, 0,002 < P < 0.02, 2-tailed Mann-Whitney Utest). Controls, which received occlusion of the left eye on day 19/20, showed the usual direction of lateralization for this task found in chicks hatched from unmanipulated eggs exposed to light; viz., impaired performance following treatment of the left hemisphere with glutamate and no effect of treating the right (U~,8 = 9.5, P = 0.009, 1-tailed Mann-Whitney U-test for the difference in the number of pecks at pebbles in the last 20 pecks, Fig. 3A). The number of pecks at pebbles in the last 20 pecks was analysed, but it can also be seen in Fig. 3 A - C that the slopes of the curves differ in a manner consistent with these scores. Unlike the data for copulation, however, occlusion of the right eye on day 1 posthatching failed to reverse the direction of lateralization for visual discrimination performance (Fig. 3C). The chicks treated in the left hemisphere showed impaired performance (Usa o = 0, P < 0.002, 2tailed Mann-Whitney U-test for the difference in the number of pecks at pebbles in the last 20 pecks). For those chicks held in the dark incubator until day 3 posthatching and then given right eye occlusion followed by glutamate treatment, there was also no reversal of the direction of lateralization. Those treated in the left hemisphere avoided pecking pebbles significantly more slowly than those treated in the right (Ug,m = 16.5, 0.02 < P < 0.05, 2-tailed Mann-Whitney U-test for the difference in the number of pecks at pebbles in the last 20 pecks; Fig. 3D). However, in this case the group treated in the left hemisphere did show some avoidance of pebbles over the testing period. It should be noted that there were no differences between any of these groups in the time taken to complete the 60 pecks.
217 A 18.
18"
LEFT EYE OCCLUDED day
RIGHT EYE OCCLUDED day E 19/20
E 19/20
16 ¸
16
14
14
12-
12 10-
O~ ,'r 1 0 . 0 EE rt" 8uJ
\
8-
\ \
6-
\
4. 2' 0 1
2
i 1
3
2
3
BLOCKS OF 20 PECKS
BLOCKS OF 20 PECKS D 18-
RIGHT EYE OCCLUDED day 1
18-
16-
16-
14 °
14.
12"
12.
RIGHT EYE OCCLUDED day 3
10-
rr
W
x\
\
0 8"
8
6'
6
\x
42-
2
0
0 1
2
3
BLOCKS OF 20 PECKS
i
I
2
3
BLOCKS OF 20 PECKS
Fig. 3. Performance on the pebble floor is presented as the mean number of errors (pecks at pebbles) in each block of 20 pecks with standard error scores. A fall in the curve indicates increasing avoidance of pebbles. The symbols are as in Fig. 2; glutamate in the left (circles), glutamate in the right (squares). Note the reversal of lateralization in those chicks which had had the right eye patched on day 19/20 of incubation (B, compared to A), but not in the groups which received this manipulation on days 1 or 3 posthatching (C and D). n = 8-10 per group.
The final procedure was aimed at finding out the number of hours of monocular light exposure, given to unmanipulated embryos, necessary to stabilise the direction oflateralization and prevent its subsequent reversal by occlusion of the right eye for 24 h on day 19/20. As shown in Fig. 4, occlusion of the right eye reversed the direction of lateralization in those embryos which had been exposed to 1 and 2.5 h of light while still in their normal position in the egg; copulation scores were elevated following treatment of the right hemisphere but not the left (Fig. 4A and B; comparison of the left and right scores using 2-tailed
Mann-Whitney U-tests gave, after 1 h of light, U8.9=6.5, 0 . 0 0 2 < P < 0 . 0 2 for d a y l 0 and U8,9 = 4.5, P < 0.002 for day 11, and after 2.5 h of light, Us,8 = 2, P = 0.000 for both days 10 and 11). In contrast, occlusion of the right eye failed to reverse the normal direction of lateralization in the group of eggs exposed to light 6 h prior to the monocular exposure. In these groups, treatment of the left hemisphere with glutamate elevated copulation, but treatment of the right did not (U8.8 = 6.5, P = 0.05 for left-right 2-tailed comparisons on days 10 and 11). The amount of light which penetrates the egg
218 t~
6~
6-
5"
0
/"
4-
F- 4 '~ i
/
//
3-
3 -
2-
6
7
8
9
10
11
6
7
AGE (DAYS)
8
9
AGE (DAYS)
10
11
6
r
e
9
'~
~1
AGE (DAYS}
Fig. 4. This figure presents data for attempted reversal of lateralization by occluding the right eye on day 19/20 of incubation following exposure of the eggs to light for 1, 2.5 or 6 h immediately prior to this manipulation. The symbols and scores are as in Fig. 2. Note the reversed direction of lateralization (opposite to that normally present) in A and B, but not C.
shell and membranes was measured at various levels of ambient illumination, and it was consistently found that only between 6% (for brown eggs) and 11% (for white eggs) of the light falling on the surface of the egg penetrates the shell and membranes. Thus, in this last experiment the eggs were exposed to between 250 and 350 lux of light and the embryos inside received in the region of 25-35 lux. DISCUSSION
The direction of functional lateralization can, as these experiments show, be reversed by occluding the right eye for 24 h while allowing the left eye to receive light stimulation. Occlusion of the right eye on day 19/20 of embryonic life has been shown to reverse the direction of lateralization for control of both copulation and performance in the pebble floor test: in these animals glutamate treatment of the right, but not the left, hemisphere elevates copulation and impairs performance in the pebble floor test. Controls, which received the same operative procedure but with occlusion of the left eye instead of the right, and so mimicked the normal eye occlusion which occurs during the later phases of development in chick embryo, exhibited the same direction of lateralization for these behaviours as found normally (i.e. that present in the brains of groups of chicks hatched from eggs which have received at
least 2 h of light stimulation during the last phases of embryonic development). They showed elevated copulation and impaired performance in the pebble floor test following glutamate treatment of the left, but not the fight, hemisphere. These results, I believe, rather conclusively confirm my original hypotheses 23 that functional lateralization in the chicken brain, for these behaviours at least, results from lateralized input of light to the eyes during the developmental period when visual connections to the forebrain are being established. Although the first connections between retinal ganglion cell axons and cells in the optic tecta begin to form as early as day 6 of embryonic life, these connections continue to develop by arborization and retraction of their axonal end terminals until around embryonic day 16 or 17, and the chick visual system is not considered to be in any way functionally mature until day 18 of embryonic development28. On day 18 of incubation an electroretinogram of embryonic form can be detected and the first behavioural responses to light stimulation can be detected. On day 19 of incubation the electroretinogram assumes its posthatching form and light-stimulated potentials can be detected for the first time in the contralateral forebrain hemisphere 11. One would therefore expect the critical period for the influence of lateralized light input on the subsequent lateralization of brain function to
219 have its onset on day 18 or 19 of incubation, although this has not been investigated experimentally. The experiments presented here were designed to determine the end point of a sensitive period for the role of lateralized light stimulation. For lateralization of performance of the pebble floor test this sensitive period ended by the time of hatching. Right eye occlusion on day 1 posthatching, and also on day 3 posthatching, is ineffective in reversing the lateralization for this behaviour in chicks incubated and hatched in darkness. In contrast, the sensitive period during which lateralized light input can influence subsequent lateralization of brain function concerned with copulation behaviour extends into posthatching life. Right eye occlusion coupled with light exposure of the left eye was found to reverse the direction of lateralization for copulation when applied on day 1 posthatching to chicks hatched in darkness from eggs incubated in darkness. Similar right eye occlusion on day 3 posthatching failed to reverse the direction of lateralization for copulation. In this case there were no overall group differences between those injected in the left and those in the right hemispheres irrespective of whether the chicks had received occlusion of their left or right eyes on day 3. Nevertheless, apparent bimodality of the individual scores in each of these groups may be taken to indicate that each individual retained lateralization for copulation although there was no common alignment amongst the members of a given :~oup (i.e. no group bias). In other words, these ,:hicks gave the same results as reported previously by Zappia and Rogers 29 for chicks ihatched from eggs incubated in darkness and brought into the light after hatching so that at no ;ime did they receive monocular light input. Thus, it would appear that by day 3 posthatching monocular, lateralized light input, be it to the left or right eye, has no effect, and the chickens behave merely as if they had not received it. The sensitive period for lateralized light input to determine the direction of lateralization for copulation has ended by day 3 posthatching, and chicks kept in darkness until this age retain lateralization, but its direction is randomly distributed amongst individuals. Therefore, in the normally developing
embryo exposed to light prior to hatching, the lateralized stimulation does not establish the presence of lateralization for copulation but, rather, aligns it in a particular direction so that all (or most) individuals in the group have a common direction of bias. The lateralization of brain function involved in performance of the pebble floor test appears to differ from this. Lateralized light input certainly plays a role in determining the direction of this form oflateralization, since right eye occlusion on day 19/20 can reverse its normal direction. Yet, rather than showing an absence of lateralization at group level, the group of chicks which received right eye occlusion on day 1, and less clearly those which received it on day 3, displayed the normal direction of lateralization. All individuals were lateralized, but in the normal direction and thus apparently uninfluenced by the right eye occlusion. One might therefore suggest that lateralization for visual discrimination learning does not require asymmetrical light input to ensure its common alignment amongst individuals in the group, but nevertheless, during the sensitive period at the end of the incubation period, its direction can be reversed by monocular, lateralized light input. This difference between the effect of light stimulation on lateralization of performance of the pebble floor test and copulation behaviour may relate to the different neural pathways involved in each, and the relative dependence of their development on genetic and environmental factors. Indeed, it has already been shown that glutamate treatment impairs performance of the pebble floor test only when its time of action is coupled with the chick viewing certain visual stimuli 27, whereas glutamate's effect in elevating copulation does not have this visual requirement 24. Copulation behaviour is well known to involve hypothalamic circuits 5 probably influenced by the forebrain 8; whereas visual discrimination performance must involve processing in either one or both of the visual systems of the forebrain (i.e. the tectorotundal-ectostriatal system and the thalamohyperstriatal system). In fact, a structural asymmetry is known to exist in the thalamo-hyperstriatal visual system of young chicks 7,25. The projections from the left side of the thalamus (fed by the
220 right eye only) to the hyperstriatum are more numerous than those from the right side of the thalamus (fed by the left eye only). This structural asymmetry is also dependent on lateralized light input as it can be reversed by occluding the right eye on day 19/20 while exposing the left to light 25. Asymmetry in these visual projections may underly the lateralization for visual discrimination performance, but it cannot be the complete explanation because the structural asymmetry is present in young males and not females 1, whereas glutamate treatment reveals functional asymmetry in both sexes. It would seem that monocular light stimulation during this sensitive period of development stimulates growth of neural projections fed either directly or indirectly by the stimulated eye. One might argue that in normal situations light received by the right eye stimulates developmental processes to occur in the left hemisphere in advance of those in the right, leading to their susceptibility to glutamate treatment later on 22. However, this may well be an oversimplified view, since each eye is not simply connected to the contralateral side of the brain, crossing over occurring in the tectal commissure and the supraoptic decussation 9. Lastly, given that as little as 2 h of light stimulation is sufficient to determine the direction of lateralization for copulation in the normal situation 21, it was considered of interest to determine the duration of light exposure necessary to prevent subsequent reversal of the lateralization by right eye occlusion. After 1 or 2.5 h of prior exposure of the eggs to light (250-350 lux at the egg's surface, but only 25-35 lux inside the egg), it was still possible to reverse the lateralization for copulation by occluding the right eye for 24 h on day 19/20 of incubation, but this could not be done after 6 h of prior exposure. That is, it takes between 2.5 and 6 h of light to stabilize the (normal) direction of lateralization so that it can no longer be reversed by this manipulation. Hence, while the sensitive period for the role of light in determining lateralization of copulation extends beyond day 1 posthatching when the eggs and hatchlings are held in darkness, monocular light exposure prior to hatching closes that sensitive period. This has similarities to the sensitive period
for imprinting, which remains open for 1 week if the chicks are dark-reared, but is closed earlier by exposure to the imprinting stimulus 17 It should be noted that it takes a shorter period of exposure to lateralized light to align the direction of lateralization (no more than 2 h) than it does to stabilise, or fix, it so that it can no longer be reversed by the converse light exposure (between 2.5 and 6 h). This makes biological sense. These experiments have demonstrated a role for non-patterned, white light on the organisation of lateralization in the forebrain, and it is perhaps surprising that an intensity as low as 25-35 lux is sufficient to bring about these effects. At least, this appears to be the intensity of light which, in these experiments, reached the embryo's right eye through the egg shell. These appear to be the first examples of rather dramatic reorganization of brain functions based on such a brief environmental exposure. Falling within a sensitive period, they have much in common with the previously demonstrated effects of visual stimulation on organization of the visual cortex in kittens 6. It should be noted that Jones and Horn 13 have reported that exposure of chicks for as little as 3 h to a flickering light on day 1 posthatching leads to increased effectiveness in synaptic transmission in the hyperstriatum, but not the ectostriatum, as measured by evoked potentials. Other experiments which have looked at the effects of monocularity in birds have involved weeks or months of monocular eye occlusion 4"16, although admittedly these studies have looked for structural or biochemical changes in the brain following monocularity rather than functional effects. Functional changes may well be more light-sensitive, but it should be remembered that they are coupled with structural reorganization of the thalamo-hyperstriatal visual projections of the brain.
ACKNOWLEDGEMENT
This research was funded by an Australian Research Council Grant A18830646.
221 REFERENCES 1 Adret, P. and Rogers, L.J., Sex difference in the visual projections of young chicks: a quantitative study of the thalamofugal pathway, Brain Res., 478 (1989) 59-73. 2 Andrew, R.J., Precocious adult behaviour in the young chick, Anita. Behav., 14 (1966) 485-500. 3 Andrew, R.J., The development of visual lateralization in the domestic chick, Behav. Brain. Res., 29 (1988) 201-209. 4 Bagnoli, P., Barsellotti, R., Pellegrini, M. and Alesc, R., Norepinephrine levels in developing pigeon brain: effect of monocular deprivation in the Wulst noradrenergic system, Dev. Brain. Res., 10 (1983) 243-250. 5 Barfield, R.J., Induction of copulatory behavior by intracranial placement of androgen in capons, Am. Zool., 4 (1964) 301. 6 Blakemore, C. and Cooper, G.F., Development of the brain depends on the visual environment, Nature (Lond.), 228 (1970) 477-478. 7 Boxer, M.I. and Stanford, D., Projections to the posterior visual hyperstriatal region of the chick: an HRP study, Exp. Brain Res., 57 (1985) 494-498. 8 Bullock, S.P. and Rogers, L.J., Glutamate-induced asymmetry in the sexual and aggressive behaviour of young chickens, Pharm. Biochem. Behav., 24 (1986) 549-554. 9 Curnod, M., Commissural pathways in interhemispheric transfer of visual information in the pigeon. In F.O. Schmitt and F.G. Worden (Eds.), The Neurosciences, Third Study Program, MIT Press, Cambridge, MA, 1973, pp. 21-29. 10 Ehrlich, D. and Saleh, C.N., The effect of eye enucleation on the number of fibres in the supraoptic decussation of posthatch chicks (Gallusgallus), Neurosci. Lett., 60 (1985) 349-355. 11 Freeman, B.M. and Vince, M.A., Development of the Avian Embryo, Chapman and Hall, London, 1974. 12 Gaston, K.E. and Gaston, M.G., Unilateral memory after binocular discrimination training: left hemisphere dominance in the chick? Brain Res., 303 (1984) 170-193. 13 Jones, S.J. and Horn, G., Effects of visual experience on photically evoked potentials recorded in the chick forebrain, Brain Res., 159 (1978) 297-306. 14 Hambley, J.W. and Rogers, L.J., Retarded learning induced by intracerebral administration of amino acids in the neonatal chick, Neuroscience, 4 (1979) 677-684. 15 Hamburger, V. and Oppenheim, R., Prehatching motility
and hatching behaviour in the chick, J. Exp. Zool., 166 (1967) 171-204. 16 Herrmann, K. and Bischoff, H.J.,Monocular deprivation affects neurone size in the ectostriatum of the zebra finch brain, Brain Res., 379 (1986) 143-146. 17 Horn, G., Memory., Imprinting and the Brain: An Inquiry into Mechanisms, Clarendon, Oxford, 1985. 18 Howard, K.J., Rogers, L.J. and Boura, A.L.A., Functional lateralization of the chick forebrain revealed by use of intracranial glutamate, Brain Res., 188 (1980) 369-382. 19 Mench, J.A. and Andrew, R.J., Lateralization of a food search task in the domestic chick, Behav. Neural Biol., 46 (1986) 107-114. 20 Rogers, L.J., Drennen, H.D. and Mark, R.F., Inhibition of memory formation in the imprinting period: irreversible action ofcycloheximide in young chickens, Brain Res., 79 (1974) 213-233. 21 Rogers, L.J., Light experience and asymmetry of brain function in chickens, Nature (Lond), 297 (1982) 223-225. 22 Rogers, L.J., Lateralization of learning in chicks, Adv. Study Behav., 16 (1986) 147-189. 23 Rogers, L.J. and Anson, J.M., Lateralization of function in the chicken forebrain, Pharm. Biochem. Behav., 10 (1979) 679-686. 24 Rogers, L.J. and Hambley, J.W., Specific and non-specific effects of neuro-excitatory amino acids on learning and other behaviours in the chicken, Behav. Brain Res., 4 (1982) 1-18. 25 Rogers, L.J. and Sink, H.S., Transient asymmetry in the projections of the rostral thalamus to the visual hyperstriatum of the chicken, and reversal of its direction by light exposure, Exp. Brain Res., 70 (1988) 378-384. 26 Rogers, L.J., Zappia, J.V. and Bullock, S.P., Testosterone and eye-brain asymmetry for copulation in chickens, Experientia, 41 (1985) 1447-1449. 27 Sdraulig, R., Rogers, L.J. and Boura, A.L.A., Glutamate and specific perceptual input interact to cause retarded learning in chicks, Physiol. Behav., 24 (1980) 493-500. 28 Thanos, S. and Bonhoeffer, F., Axonal arborization in the developing chick retinotectal system, J. Camp. Neurol., 261 (1987) 155-164. 29 Zappia, J.V. and Rogers, L.J., Light experience during development affects asymmetry of forebrain function in chickens, Dev. Brain Res., 11 (1983) 93-106. 30 Zappia, J.V. and Rogers, L.J., Sex differences and reversal of brain asymmetry by testosterone in chickens, Behav. Brain Res., 23 (1987) 261-267.