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Exposure to different wavelengths of light and the development of structural and functional asymmetries in the chicken
ELSEVIER
BEHAVIOURAL BRAIN RESEARCH Behavioural Brain Research 80 (1996) 65-73
Research report
Exposure to different wavelengths of light and the development of structural and functional asymmetries in the chicken L.J. R o g e r s *, G . A . K r e b s
Department of Physiology, University of New England, Armidale, N.S.W. 2351, Australia Received 11 July 1995; revised 16 January 1996; accepted 22 January 1996
Abstract The thalamofugal visual projections of the chick are known to develop in response to stimulation by light prior to hatching, and asymmetry in the number of projections develops as a consequence of the embryo being oriented in the egg so that it occludes its left eye. The right eye only is stimulated by light and this causes the visual projections connected to the right eye to develop in advance of those connected to the left. We have now found that exposure of embryos, from day 19 of incubation to hatching, to red (peak transmission at 670 nm) or green (peak at 500 nm) light is as effective as broad-spectrum (white) light in establishing asymmetry in these projections. The intensities of the light to which the embryos were exposed in each case were equivalent, achieved in part by removing the air sac end of the egg shell. The thalamofugal visual projections, therefore, develop in response to light stimulation but appear to have no wavelength specificity. This result is consistent with the apparent lack of involvement of the thalamofugal visual pathway in colour vision. However, functional asymmetry, tested as left-right eye differences in categorising grain from pebbles, was found to be less marked in the chicks that had been exposed to green light compared to those that had been exposed to 'white' light, and it was absent in those exposed to red light or incubated in the dark. Thus, there is wavelength specificity for the development of the behavioural asymmetry, which suggests involvement of colour-coded neurons outside the thalamofugal visual pathway, probably in the tectofugal pathway. Exposure of the embryos to red and green light alternating at 30 min intervals is as effective as 'white' light for establishing both the structural and functional asymmetry.
Keywords: Thalamofugal projections; Development; Wavelength specificity;Asymmetry;Visual behaviour
1. Introduction Birds have two main visual pathways, one which projects from the tectum to the ectostriatal region of the forebrain and the other which projects from the thalamus to the visual Wulst or hyperstriatum 1-18]; see Fig. 1. Structural asymmetries are present in both of these visual pathways [36]. Giinttlrk~in et al. [14] have shown that there is asymmetry in the tectofugal visual pathway of the pigeon. In layers 10-15 of the optic tectum the cells are larger and have more dendrites on the right side. A more recent study 1-22] has shown that the larger cell size on the right side is confined to layer 13 of the rectum and another study has shown that the cells in layers 2-7 of the tectum are larger on the left side [ 12]. *Corresponding author. Fax: +61-67-73-3234; E-mail: [email protected]. 0166-4328/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S016-64328 (96)00021-6
The cells in layers 2 - 7 receive input from the retina and they project to layer 13. The cells in layer 13 of the tectum receive input from the tectal commissures, the hyperstriatum and the archistriatum, and they project to the nucleus rotundus [6]. Their larger size on the right side may indicate more input from the contralateral tectum than their counterparts on the left side and also that they give rise to more projections to the rotundal nuclei. In fact, GOnt~irkOn and Melsbach [13] have reported that the left nucleus rotundus receives afferents from both sides of the optic tectum, with only a slightly greater bias for more from the ipsilateral tectum, whereas the right nucleus rotundus receives afferents almost exclusively from the right rectum. That is, inputs from the right eye are relayed firstly to the left tectum and from there almost exclusively to the left nucleus rotundus, whereas inputs from the left eye are relayed to the right tectum and from there to both rotundal nuclei. It is known that there are bilateral projections to the
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Fig. 1. The two visual pathways in the avian brain are illustrated in a simplifiedform. Note that the thalamofugal visual projections,arising from a collectionof nuclei in the thalamus known as the opticus principalis (OPT), feed forward to the hyperstriata on both sides of the forebrain.The contralateral projections are indicated by the grey lines. Note that input from the eye (grey lines) goes to the contralateral OPT and the contralateral tectum opticum (TO). The ipsilateral projections are indicated by the black lines. The stippled area in the hyperstriatum (HA and HD) indicates the approximate site of the dye injection and its spread from that site. Other abbreviations: Hp, hippocampus; HV, hyperstriatum ventrale; ROT, nucleus rotundus; E, ectostriatum;N, neostriatum; HA, hyperstriatum accessorium;HD, hyperstriatum dorsale.
rotundal nuclei in chicks [24], as in the pigeon, but so far there has been no investigation of asymmetry in these projections in chicks. Asymmetry in the thalamofugal visual pathway has been shown in the chicken [1,42]. The left side of the thalamus, which receives input from the right eye only, sends a larger number of projections to the hyperstriaturn than does the right side of the thalamus, which receives input from the left eye only. This asymmetry in the thalamofugal projections was first reported by Boxer and Stanford [5], who injected horseradish peroxidase into the hyperstriatum of 8-day-old chicks and located the asymmetry in the contralateral projections from either side of the thalamus to the hyperstriatum. This result has been confirmed by injection of retrograde labelling fluorescent dyes on day 2 or day 12 of posthatching life [1,42,44]. The above technique has revealed that the asymmetry is present in both males and females, but it is greater in males, at least on day 2 post-hatching [27]. The asymmetry of the visual projections is consistent with the lateralization of function present in young chicks [3,10,37]. For example, when tested monocularly, chicks using the right eye perform better in visual discrimination tasks [10,23,51]. Chicks using the left eye are more attentive to topographical cues and respond to small changes in the stimuli, whereas chicks using the right eye pay more attention to colour and respond only to large changes in the stimuli [2,28,34,45]. Although asymmetry on visual tasks has been reported to occur
in both males and females [23], in some studies females show a lesser degree of asymmetry than males [3,51]. The lateralization of the visual projections and of visual function in the chick has been shown to depend on exposure of the embryo to light during the last 3 to 4 days prior to hatching. During this stage of development, the embryo is oriented in the egg so that the right eye is positioned next to the air sac and the left eye is occluded by the body [31]. Thus, only the right eye receives stimulation by light entering through the shell and membranes. Chickens hatched from eggs that have been exposed to two or more hours of broad-spectrum, incandescent or fluorescent light on day 19/20 of incubation show lateralization for control of visual discrimination performance, whereas chickens hatched from eggs incubated in the dark show no asymmetry [32,40,50]. Furthermore, it is possible to reverse these asymmetries by withdrawing the embryo's head from the egg, occluding the right eye and allowing the left eye to receive stimulation by light [33]. The structural asymmetry in the visual projections is affected by stimulation by light in a similar manner. Exposure of the embryo's left eye to light reverses the direction of the asymmetry in the thalamofugal visual projections [42] and no asymmetry is present in chicks incubated in darkness [40]. Thus, stimulation by light prior to hatching influences the growth of the visual neurons which project from the thalamus to the forebrain [36]. The same dependence on light stimulation occurs for the development asymmetry in the tectofugal path-
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way: pigeons hatched from eggs incubated in darkness have no asymmetry of cell size [ 12]. It should be noted that the pigeon is an altricial species, hatching at an earlier state of development compared to the chicken. The earlier effect of light exposure on asymmetry in the tectofugal pathway may reflect its apparent earlier development compared to the thalamofugal visual pathway, as known to occur in chicks [38,39]. Another interesting comparison between the two visual pathways in birds is the involvement of the tectofugal pathway, but not the thalamofugal pathway, in colour vision [15,26,46]. Whereas the two pathways have different functional roles for colour vision, it is not known whether the same differentiation occurs for their light-stimulated development. Development of the tectofugal pathway might depend on wavelength, whereas development of the thalamofugal might not. We were, therefore, interested to see whether colour is a parameter of the light stimulation which generates the known structural and functional asymmetries in chicks, and we tested this by exposing embryos to different wavelengths of light.
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One control group was exposed to the broad-spectrum incandescent light (without filtering) and another was incubated in darkness. (From here on, the groups will be referred to as green-light exposed, red-light exposed, alternating-light exposed, white-light exposed and darkincubated.) The term 'white-light' will be used to describe the broad-spectrum produced by the incandescent bulbs although it is recognised that they produce wavelengths largely over the red to green region of the spectrum and have relatively little output of the shorter wavelengths. The eggs were left undisturbed from day 19 of incubation until hatching. After hatching, the chicks were housed in groups of 2 to 3, and supplied food and water ad libitum. The home cages were fitted with clear perspex fronts (22 cm × 22 cm x 30 cm high). Warmth and light were provided by overhead 40 W incandescent bulbs (approximately 1000 lux). The cages were cleaned daily. The chicks were sexed either after they had been tested behaviourally or at the time of perfusion for histology. The behavioural data for males only is reported here, and histology was performed on the brains of males only. This choice was made because males are known to have a greater degree of lateralization than females [3,23,27,51].
2. Materials and methods
2.1. Animals and housing conditions White Leghorn × New Hampshire eggs were incubated in darkness until day 19 of incubation in a forceddraught, automatically turning incubator. On day 8 the eggs were candled using white light. On day 19, the eggs were divided into five equal groups. The egg shell was removed from the air sac end of the egg exposing the membranes over the head of the embryo. This procedure prevented the shell from impeding light input to the right eye and allowed precise control over the exposure to different wavelengths of light, as the wavelengths used might be filtered as they pass through the shell. After removal of the shell, the eggs were moved to Multiplo incubators which had been adapted for heating with an element rather than a light bulb. Light exposure of the eggs was achieved by placing incandescent lamps (40 W bulbs) over windows in the lid of the incubators. The intensity of light was adjusted using a rheostat so that the four groups of eggs were exposed to approximately 400 lux of light after filtering (measured using a Topcon Digital Illuminance Meter Model IM-3 placed alongside the eggs). One group was exposed to red light (incandescent 40 W bulbs filtered by a Rosco Supergel number 27 with transmission wavelength ranging from 620 to above 740 nm and a peak transmission at 670 nm; Fig. 2), another to green light (Rosco Supergel number 75 transmitting over the range of 460-580 nm with a peak at 500 nm; Fig. 2), and a third group was exposed to red and green light alternating at 30-min intervals.
2.2. Behavioural test for lateralization of brain function The ability to categorise grains of chick mash as distinct from pebbles was tested in a standard pebblefloor test requiring the chicks to search for grains of chick starter crumbles scattered on a background of pebbles [43]. The chicks were tested monocularly on day 8, following occlusion of one eye by a conicalshaped piece of adhesive tape applied 5 min before testing. The number of pecks at pebbles and grains were scored for a total of 60 pecks. Prior to testing, the chicks were deprived of food for 3 h. Only pecks involving new choices of grain or pebbles were scored. Error scores (pecks at pebbles) were recorded for each block of 20 pecks. The number of errors in the last block of 20 pecks (pecks 41-60) were the standard used to determine functional lateralization (for more detail see [34]). 2.3. Labelling the thalamofugal visual projections with fluorescent dyes
To determine the number of projections from each side of the thalamus to the forebrain, the retrogradely transported fluorescent dyes True Blue (Sigma T5891) and Fluorogold (Fluorochrome Inc.) were injected into the hyperstriatum, one into each side of the forebrain. The chicks to be injected with dye were taken from the same batches of eggs as were the chicks tested behaviourally, apart from the control groups incubated in darkness. The absence of asymmetry in chicks incubated in
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~
0.18
100
0.16
Wavelength (nm) Fig. 2. The percentage transmission spectra of the two filters is presented (dotted area for green filter and grey area for red filter, see text for details). The dashed line presents the absorbance spectrum for rod cells in the chick retina, as reported by Bowmaker and Knowles I-4]. The black arrow heads along the horizontal axis represent the positions of maximal absorption of the various cone cell pigments 1-47]. The white arrow heads indicate shifts that are calculated to occur due to the presence of red and yellow oil droplets [47J.
darkness and raised in identical conditions to those mentioned above has been reported previously [40]. The method of dye injection has been described previously in detail [ 1,40]. Briefly, on day 2 post-hatching, the chicks were anaesthetized with an intramuscular injection of 50 mg/kg ketamine and 5 mg/kg xylazine and placed in a stereotaxic apparatus. Using a Hamilton syringe, 0.6 #1 of a 5% solution of True Blue was then injected into the hyperstriatum aceessorium region of one of the hemispheres and 0.4 pl of a 4% solution of Fluorogold was injected into the same region in the other hemisphere. These different doses were used with the aim of obtaining approximately equal numbers of labelled neurons with each dye. The side into which each dye was injected was randomly assigned to control for any effects particular to each dye. After recovery, the chicks were returned to their home cages until they were 6 days old, when they were deeply anaesthetized with sodium pentobarbital and perfused with a solution of 10% formalin in 0.1 M phosphate buffer. The chicks were sexed by inspection of the gonads and the brains of the males were removed and sectioned into 40-/~mthick slices using a freezing microtome, with alternate sections mounted immediately onto gelatine-subbed slides. The sections were then dried and the slides were coverslipped. Specific regions in the forebrain and thalamus were identified with the aid of a stereotaxic atlas [19]. Inspection of the injection site in the hyperstriatum and of labelled cells in the thalamus was performed using ultraviolet microscopy. Injection site parameters for each dye were determined by examining sections of the fore-
brain. The distance from the rostral aspect of the brain to the section containing the greatest area of dye around the injection site, presumed to be the centre of the injection site, was determined. With the aid of an ocular micrometer grid, the distances from the centre of fluorescence to the midline of the brain and to the dorsal surface of the brain were measured on this section. The diameter of the area of fluorescence in this section was also measured to estimate the size of the injection site. Previous studies have shown that the region of spread of the dye around the injection site approximates a sphere [40,41 ], and hence the volume of this region was calculated from the measured diameter. From thalamic sections of each brain, the number of labelled cells ipsilateral and eontralateral to the injection site were determined, using the previously described method [40,41]. The total number of cells in the side of the thalamus contralateral (C) to the injection site was divided by the total number of labelled cells in the side of the thalamus ipsilateral (I) to the injection site to determine the C/I ratio for each dye/hemisphere. Use of this ratio controls for variations in the amount of dye injected [ 1].
2.4. Analysis of data The behavioural data was analysed using a generalised linear model with binomial error function and LOGIT LINK FUNCTION. Injection sites and C/I ratios were compared using two-factor ANOVAs and Pearson correlation. Post-hoe t-tests were applied, one-tailed only when the result could be predicted from previous studies.
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3. Results 3.1. Behavioural test In the pebble-floor test, there were significant main effects of light treatment (F4,84 = 3.10, 0.01 < P < 0.025) and of the eye used during testing (F1,84=70.93, P<0.001); see Fig. 3A. There was also a significant interaction between the light treatment and the eye used (F4,84 = 4.81, 0.001 < P < 0.005). As expected from previous studies, chicks exposed to white-light and tested using the right eye made significantly fewer pecks at pebbles in the last 20 pecks than those using the left eye (ts4=6.46, P<0.001). This was also the case for chicks that were exposed to green light, and to alternating red and green light (t84=3.79, P<0.002; and t84=4.56, P<0.002, respectively). However, in chicks
A ~
-v
16
14
69
exposed to red light there was no significant difference in performance tested using the left or right eye (ts4= 1.93, 0 . 1 0 < P < 0 . 2 0 ) , and the same was true in the dark-incubated chicks (ts4 = 1.53, 0.20 < P < 0.40). The degree of lateralization was greatest in white-light exposed chicks. Chicks that were exposed to green light during incubation and were tested using the right eye made significantly more pecks at pebbles than whitelight exposed chicks using the right eye (ts4=3.47, P<0.002). This also applies to chicks that were redlight exposed when they were tested using the right eye (t84=2.90, 0.002
"T"
3.2. Labelled visual projections
B
iil
*L
Y__
1 DARK
WH1TE
RED
GREEN
FLASH R/G
Fig. 3. Lateralization in behaviour and in the structure of the thalamofugal projections. A: The number of pecks at grains (as opposed to pebbles) in the last 20 pecks of the pebble-floortest in chicks tested monocularly using the left eye (black bars) or fight eye (white bars). B: C/I ratios (see text) followinginjection of tracer dyes into the left byperstriatum (black bars) or right hyperstriatum (white bars). Means and S.E.M.s are presented and asterisks represent significant left-right differences (P<0.05). The data are for chicks exposed to the various light conditions prior to hatching. Note that data for C/I ratios followingdark incubation (grey and dotted bars) are from a previous study [40].
Examination of the injection sites in the forebrain confirmed that the centres of all of the injection sites were located in the hyperstriatum accessorium (HA) and that injected dye had diffused into the region of the hyperstriatum dorsale (HD) and the nucleus intercalatus of the HA (IHA), the regions in which thalamofugal projections terminate [18]. The site of the dye injection was at a mean depth___S.E.M, of 1.38+_0.06mm from the dorsal surface of the brain. As in earlier studies [ 1,40], for both Fluorogold and True Blue, no significant correlation was found between the volume of the injection site in the forebrain and the C/I ratio ( Pearson correlation, r=0.13). The mean volume of the injection site was 1.03 +0.17 mm 3 for Fluorogold and 0.81 +0.18 mm a for True Blue. Analyses of variance indicated that the values for depth of injection and volume of the dye injected did not differ significantly among the four treatment groups or between the left and right hemispheres. ANOVAs of the data for the rostral displacement (1.11 ___0.04 mm) and lateral displacement ( 1.05 _+0.06 mm) did not show a main effect of left/right side (F3,42=0.006, P = 0 . 9 4 and F3,42=0.0003, P=0.99, respectively). However, the rostral and lateral displacements of the injection sites were found to differ significantly between the light treatments (F3.~2=3.00, P = 0.04, and F3,42=4.28, P - 0 . 0 1 , respectively), although on both sides injection sites were confined to the HA. This unintentional error in the placement of injections does not appear to have affected the values of the C/1 ratios, since no significant correlation was found between
L.J. Rogers, G.A. Krebs/Behavioural Brain Research 80 (1996) 65- 73
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the C/I ratios and either the distance of the injection site from the midline (r= 0.3) or the rostral displacement
4. Discussion
(r = 0.09).
Asymmetry in the organisation of the thalamofugal visual projections of the young chick is known to develop following exposure of the embryo to broad-spectrum light [ 32, 36, 50] and the present results confirm this fact. Previous research indicates that the asymmetry is present in the contralateral projections [5,40] and, thus, there are more projections from the left thalamus to right hyperstriatum than from the right thalamus to left hyperstriatum. Here we have shown that stimulation of the right eye of the embryo by red or green light of equivalent intensity to the 'white' (or broad-spectrum) light generates the same asymmetry in 2-day-old chicks. This result should be compared to the previously reported absence of asymmetry in the thalamofugal visual projections of chicks hatched from eggs incubated in darkness [40] (see also Fig. 3B). The apparent lack of wavelength specificity for light-dependent development of the thalamofugal visual projections is consistent with the evidence that lesions of the hyperstriatum have little effect on colour vision [26] compared to lesions placed in the tectofugal visual pathway [15,25]. The role of the tectofugal pathway in colour vision is supported by the fact that cells in the dorsal-anterior region of the rotundal nuclei of the tectofugal pathway respond vigorously to specific colours [48]. We recognise that the experiments reported here need to be repeated with exposure of the embryo to other wavelengths of light before a firm conclusion can be reached, but it appears that the asymmetrical development and lateralized function of the thalamofugal visual pathway lacks wavelength specificity. The fact that exposure to alternating red and green light generates no greater asymmetry than exposure to either the red or green light alone indicates that both colours of light may influence the development of the same population of neurons. That is, the lack of an additive effect of the alternating red and green light indicates that there may not be separate populations of neurons that develop in response to stimulation by either red or green light. Indeed, there was an indication that the chicks exposed to the alternating red and green light might have a lesser degree of asymmetry than any of the other groups but this was not significant. Of course, it might be argued that these chicks received insufficient exposure to either colour of light to promote the development of asymmetry, but the total amount of exposure to each colour exceeded 24 h, a sufficient period of exposure for broadspectrum light to cause structural asymmetry [42]. We also investigated the effects of exposing the embryo to the different colours of light on the lateralization of visually guided behaviour tested on day 8 post-hatching. It is known that exposure of the embryo to white light on day 19 of incubation for as little as 2h leads to lateralization of performance on the task requiring the
Labelled cells were confined to the dorsolateral anterior thalamus (DLA) (rostrolateral and lateral parts), in the region of the nucleus opticus principalis thalami (nOPT), a region in the thalamofugal pathway which receives direct input from the retinae and projects to the HD and IHA [7,181. Photographs and detailed diagrammatic representations of both the projection sites in the forebrain and the labelled cells in the thalamus have been published previously [ 1,42]. The C/I ratios were analysed by a 2-way ANOVA (light xside). No main effect of light was found (F3,42 = 1.31, P=0.28), but there was a significant main effect of the side injected (F1.42= 14.98, P=0.0004). There was no significant interaction ( F 3 , 4 2 = 0 . 3 4 , P = 0.80). Thus, all of the light conditions produced asymmetry (see Fig. 3B). Since the two hemispheres of each brain were injected with different dyes, left and right C/I ratios of individuals could be compared using paired statistical tests. In the group of chicks exposed to white-light, the mean C/I ratio following injection of the right hemisphere was significantly higher than that following injection of the left hemisphere (Fig. 3B; one-tailed paired t-test, P = 0.001). This asymmetry is in agreement with previous studies of untreated, light-incubated male chicks [1,40,42]. Structural asymmetry was found also in the chicks exposed to red, green and alternating red and green light (paired t-tests, P=0.018, P=0.02, and P = 0.04, respectively). Comparison of the left-right differences in the C/I ratio calculated as ( R - L / R + L) values revealed that the same degree of asymmetry was present in all groups (F3.22 = 1.1, P=0.37; Fig. 4).
0,4-
0.3-
02-
lil l iii!i
0.1-
WHITE
RED
iiiiiiii !ill GREEN FLASHR/G
Fig. 4. The C/I ratios for injections into the left and right sides of the hyperstriatum have been expressed as ( R - L / R +L) for each individual. This figure presents the mean values for each group with S.E.M.s. There is no significant difference between any of the groups, although there is a trend for less asymmetry to be present in the thalamofugal visual system of the group exposed to alternating red and green light (Flash R/G).
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chick to shift from pecking randomly at pebbles and grain to pecking at grain only [33]. This lateralization has been shown both by unilateral treatment of the forebrain hemispheres with either cycloheximide or glutamate and by monocular testing [30,35]. Drug treatment of the left, but not the right, hemisphere impairs performance on this task [17,37] and occlusion of the right, but not the left, eye produces the same impairment [23,51]. Thus, the task is best performed when the chick uses the right eye and the left hemisphere. The results reported here show that this advantage of the right eye and the left hemisphere does not develop if the embryos are incubated in darkness. Although following incubation in the dark there was a slight tendency for the group of chicks using the right eye to perform better than the group using the left eye, this difference was not significant. In fact, in the chicks incubated in darkness, use of neither the left or right eye allowed the chick to shift away from pecking randomly at pebbles and grain. This result correlates with an absence of asymmetry in the thalamofugal visual projections of chicks incubated in darkness [40]. Exposure of the embryos to red light failed to establish lateralization of performance on the pebble-floor task, whereas exposure to green light did produce significant lateralization. Therefore, the development of this particular functional asymmetry is wavelength dependent. Red light was clearly less effective than green light. The chicken has cone pigments which give rise to three peaks of maximal absorption between 400-500 nm, another at about 500 nm and yet another around 570 nm [4,21,46,47]; see Fig. 2. The rods have an absorbance maximum at 506 nm and a cut off around 600 nm in the longer wavelength direction [4]; see Fig. 2. Thus, the red filter that we used would stimulate cones and not rods, whereas the green filter would stimulate both rods and cones. In fact, the transmission range of the green filter overlaps almost exactly the absorbance spectrum of the rods. The lesser effect of exposure to red light, compared to green, in establishing the behavioural asymmetry might possibly depend on a lack of stimulation of rod cells in the retina although the wavelength specificity might be explained equally well by selective stimulation of the different populations of cone cells. Judging from results obtained with pigeons, the behavioural ability to discriminate between different wavelengths is better for the shorter wavelengths, which includes the green region of spectrum, than for the longer wavelengths above 600 nm [46]. Better ability to discriminate in the green region of the spectrum is also consistent with the known absorbance spectrum for the retinal photoreceptors and oil droplets [4]. Therefore, the wavelengths that we have found to be most effective for stimulating development of the visual pathways can also be discriminated best by the bird. It should, however, be noted that Wang et al. [48] have found in
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pigeons that most of the colour-coded cells in the dorsalanterior part of the nucleus rotundus are more sensitive to wavelengths in the red and the blue regions of the spectrum, with low responsiveness to green. This apparent contradiction cannot be reconciled with the behavioural studies, nor with our results. Our finding that exposure of the embryo to red light leads to asymmetry in the thalamofugal visual projections but not lateralization of performance on the pebblefloor task suggests the thalamofugal visual pathway may have either no role or a minor role relative to the tectofugal visual pathway in this particular visually guided behaviour. Previously, one of the authors [30] suggested that performance on the pebble-floor task relies primarily on use of the thalamofugal pathway, based on the age-dependent loss of functional asymmetry in parallel with the loss of asymmetry in the thalamofugal pathway by day 21 post-hatching. This conclusion would now seem to be incorrect. Moreover, the binocular frontal field of vision is used for pecking in the pebblefloor task and it is known to be exclusively tectofugal in the pigeon [14]. As a chick searching for grain on the pebble-floor task uses both the frontal and lateral visual fields, it is likely that both visual pathways are used in performing this task. There is also evidence for communication between the two visual pathways via projections from HA first to the hyperstriatum ventrale and then to the peri-ectostriatal belt and also between HA and the optic tectum [8,9]. The function of the thalamofugal visual pathway remains unclear. The tectofugal visual pathway is known to be involved in colour vision, visual acuity, detection of movement and pattern discrimination [11,29,48], whereas the thalamofugal visual pathway may be involved in pattern discrimination and detection of movement, but not in acuity or, as mentioned above, in colour vision [14,20,49]. There is some evidence that the processing of thalamofugal inputs to the forebrain enhances performance on visual discrimination tasks, but that the tectofugal inputs provide the essential processing [16]. In the task used in this study, the grains cannot be distinguished from pebbles on the basis of colour, size or shape. They can be distinguished by differences in texture and brightness and, because the grain can be moved by either scratching or pecking with a closed beak, they might also be distinguished by movement relative to the background. Both visual pathways might contribute to performance on this task, but it is likely that the acuity abilities of the tectofugal pathway play an essential part in guiding the shift to pecking almost exclusively at grain. If so, our results suggest that asymmetry might be established in the tectofugal pathway by exposing the embryo to green, but not red, light. This would explain the dissociation between asymmetry in the thalamofugal pathway, caused by red light, and the lateralized performance of the
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pebble-floor task. Moreover, wavelength specificity for the development of the tectofugal pathway would be consistent with the clear colour specificity of neurons within the nucleus rotundus [48]. Alternatively, the asymmetry generated by exposing the embryo to green, but not red, light and manifested in the behavioural task may occur at a higher level of cognitive integration, outside the primary visual pathways. Exposure of the embryos to alternating red and green light also produced significant lateralization of performance on the pebble-floor task. Unless red light can synergise with green light, even though it has no effect on its own, the lateralization must be caused by the exposure to green light for half of the entire period used in this experiments (i.e. for about 24 h). It would now be interesting to know whether, as for broad-spectrum light, as little as 2 h of green light is sufficient to generate the lateralization. These results show clear effects of light of different wavelengths on the development of functional and structural asymmetries in the chick. It appears that exposure of the embryo to green light may generate asymmetry in both of the visual pathways, whereas exposure to red light may do so only in the thalamofugal visual pathway. In this study we have tried to establish associations between asymmetry in a neural pathway and behaviour, and to draw links between the effects of sensory stimulation on the development and the functional specificity of these neurons. The thalamofugal visual projections appear to lack wavelength specificity both for stimulation of their development and for their later use in visual perception.
Acknowledgement This research was supported by an ARC grant number A09130918 to L.J. Rogers. We thank to Dr. A.N.B. Johnston for helpful suggestions.
References I-1] Adret, P. and Rogers, L.J., Sex differences in the visual projections of the young chick: a quantitative study of the thalamofugal pathway, Brain Res., 478 (1989) 59-73. [2] Andrew, R.J., The development of visual lateralization in the domestic chick, Behav. Brain Res., 29 (1988) 201-209. 1-3 ] Andrew, R.J. and Brennan, A., Sex differences in lateralization in the domestic chick, Neuropsychologia, 22 (1984) 503-509. [4] Bowmaker, J.K. and Knowles, A., The visual pigments and oil droplets of the chicken retina, Vision Res., 17 (1977) 755-764. 1-5] Boxer, M.I. and Stanford, D., Projections to the posterior visual hyperstriatal region in the chick: an HRP study, Exp. Brain Res., 57 (1985) 494-498. [6] Curnod, M. and Streit, P., Amino acid transmitters and local circuitry in the optic tectum. In F.O. Schmidt and F.G. Warden
(Eds.) The Neurosciences, MIT Press, Cambridge, MA, 1980, pp. 989-1004. [7] Ehrlich, D. and Mark, R., An atlas of primary visual projections in the brain of the chick Gallus gallus, J. Comp. Neurol., 223 (1984) 592-610. [ 8] Engelage, J. and Bischof, H-J., The organization of the tectofugal pathway in birds: A comparative review. In H.P. Zeigler and H-J. Bischof (Eds.), Vision, Brain, and Behavior in Birds, MIT Press, Cambridge, MA, 1993, pp. 137-158. I-9] Engelage, J. and Bischof, H-J., Visual wulst influences on flash evoked responses in the ectostriatum of the zebra finch, Brain Res., 652 (1994) 17-27. [10] Gaston, K.E. and Gaston, M.G., Unilateral memory after binocular discrimination training: left hemisphere dominance in the chick, Brain Res., 303 (1984) 190-193. [11] Granada, A.M. and Yazulla, S., The spectral sensitivity of single units in the nucleus rotundus in pigeons, Brain, Behav. Evol., 2 (1971) 363-384. [12] Gtintt~rkiin, O. The ontogeny of visual lateralization in pigeons, German J. Psychol., 17 (1993) 276-287. [13] Gtint0rkian, O. and Melsbach, G., Asymmetric tecto-rotundal projections in pigeons: Cues to the origin of lateralized binocular integration. In N. Eisner and D.W. Richter (Eds.), Rhythmogenesis in Neurons and Networks: Proceedings of the 20th Grttingen Neurobiologentagung, Thiem, Stuttgart, 1992, p. 364. [14] GfintiirkOn, O., Emmerton, J. and Delius, J.D., Neural asymmetries and visual behaviour in birds. In H.C. Liittgau and R. Necker (Eds.), Biological Signal Processing, VCH VerlagsgeseUschaft; Weinheim: FRG, Deutsche Forschungsgemeinschaft, 1989, pp. 122-145. [15] Hodos, W., Color discrimination deficits after lesions of nucleus rotundus in pigeons, Brain Behav. Evol., 2 (1969) 185-200. [ 16] Hodos, W., The visual capabilities of birds. In H.P. Zeigler and H.J. Bischof (Eds.), Vision, Brain, and Behaviour in Birds, MIT Press, Cambridge, MA, 1993, pp. 63-76. [ 17] Howard, K.J., Rogers, L.J. and Boura, A.L.A. Functional lateralization of the chicken forebrain revealed by use of intracranial glutamate, Brain Res., 188 (1980) 369-382. [18] Karten, H.J., Hodos, W., Nauta, W.J.H. and Revzin, A.M. Neural connections of the 'visual Wulst' of the avian telencephalon. Experimental studies in the pigeon Columba livia and the owl Speotyto eanicularia, J. Comp. Neurol., 150 (1973) 253-276. [ 19] Kuenzel, W.J. and Mason, M., A Stereotaxic Atlas of the Brain of the Chicken (Gallus gallus), John Hopkins University Press, Baltimore, 1988, 166 pp. [20] Mack, K.A. and Hodos, W., Near-field acuity after visual system lesions in pigeons: I. Thalamus, Behav. Brain Res., 13 (1984) 1-14. [21] Maier, E.J. and Bowmaker, J.K., Colour vision in the passiform bird, Leiothrix lutea: correlation of visual pigment absorbance and oil droplet transmission with spectral sensitivity, J. Comp. Physiol. A, 172 (1993) 295-301. [22] Melsbach, G., Hahmann, U., Waldmann, C., WOrtwein, G. and Gtintiirk~in, O., Morphological asymmetries of the optic tectum of the pigeon. In N. Eisner and H. Penzlin (Eds.), Synapse-Transmission-Modulation, Thiem, Stuttgart, 1991, p. 553. [23] Mench, J.A. and Andrew, R.J., Lateralization of a food search task in the domestic chick, Behav. Neural Biol., 46 (1986) 107-114. [24] Ngo, T.D., Davies, D.C., Egedi, G.Y. and TOmbrli, T., A phaseolus lectin anterograde tracing study of the tectorotundal projections in the domestic chick, J. Anat., 184 (1994) 129-136. [25] Palacios, A., Gianni, H. and Varela, F., Chromatic discrimination in pigeons after thalamic lesions of nuclei Rotundus (Rt) and Geniculatus Lateralis ventralis (GLv): A psychophysical study, C. R. Acad. Sci. Paris, 312 (III) (1991) 113-116. [26] Pritz, M.B., Mead, W.R. and Northcutt, R.G., The effects of Wulst ablations on color, brightness and pattern discrimination in pigeon (Columba livia), J. Comp. Neurol., 140 (1970) 81 100.
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[27] Rajendra, S. and Rogers, L.J., Asymmetry is present in the thalamofugal visual projections of female chicks, Exp. Brain Res., 92 (1993) 542-544. [28] Rashid, N. and Andrew, R.J., Right hemisphere advantage for topographical orientation in the domestic chick, Neuropsychologia, 27 (1989) 937-948. [29] Revzin, A.M., Functional localization in the nucleus rotundus. In A.M. Granada and J.H. Maxwell (Eds.), Neural Mechanisms of Behaviour in the Pigeon, Plenum, New York, 1979, pp. 165-176. [30] Rogers, L.J., Development of lateralisation. In R.J Andrew (Ed.), Neural and Behavioural Plasticity: The Use of the Domestic Chick as a Model, Oxford University Press, Oxford, 1991, pp. 507-535. 1-31] Rogers, L.J., Lateralization of learning in chicks, Adv. Study Behav., 16 (1986) 147-189. 1-32] Rogers, L.J., Light experience and asymmetry of brain function in chickens, Nature, 297 (1982) 223-225. [ 33 ] Rogers, L.J., Light input and the reversal of functional lateralisation in the chicken brain, Behav. Brain Res., 38 (1990) 211-221. [34] Rogers, L.J., The Development of Brain and Behaviour in the Chicken, CAB International, Wallingford, 1995, pp. 273. [ 35] Rogers, L.J., The molecular neurobiology of early learning, development, and sensitive periods, with emphasis on the avian brain, Mol. Neurobiol., 7 (1993) 161 187. 1,36] Rogers, L.J., Behavioral, structural and neurochemical asymmetries in the avian brain: a model system for studying visual development and processing, Neurosci. Biobehav. Rev., (1996) in press. [37] Rogers, L.J. and Anson, J.M., Lateralization of function in the chicken forebrain, Pharm. Biochem. Behav., 10 (1979) 679-686. 1,38] Rogers, L.J. and Bell, G.A., Changes in metabolic activity in the hyperstriatum of the chick before and after hatching, Int. J. Dev. Neurosci., 12 (1994) 557 566. 1,39] Rogers, L.J. and Bell, G.A., Different rates of functional development in the two visual systems of the chicken revealed by [14C]2-deoxyglucose, Dev. Brain Res., 49 (1989) 161-172. [40] Rogers, L.J. and Bolden, S.W., Light-dependent development and
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asymmetry of the visual projections, Neurosci. Lett., 121 (1991) 63-67. [41] Rogers, L.J. and Rajendra, S., Modulation of the development of light-initiated asymmetry in the chick thalamofugal visual projections by oestradiol, Exp. Brain Res., 93 (1993) 89-94. [42] 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. [43] Rogers, L.J., Drennen, H.D. and Mark R.F., Inhibition of memory formation in the imprinting period: irreversible action of cycloheximide in young chicks, Brain Res., 79 (1974) 213-233. [44] Schwarz, I.M. and Rogers, L.J., Testosterone: A role in the development of brain asymmetry in the chick, Neurosci. Lett., 146 (1992) 167-170. [45] Vallortigara, G. and Andrew, R.J., Lateralization of response by chicks to change in model partner, Anita. Behav., 41 (1991} 187-194. [46] Varela, F.J., Letelier, J.C., Marin, G. and Maturana, H.R., The neurophysiology of avian colour vision, Arch. Biol. Med. Exp., 16 (1983) 291-303. 1,47] Varela, F.J., Palacios, A.G. and Goldsmith, T.H., Color vision of birds. In H.P. Zeigler and H.-J. Bischof (Eds.), Vision, Brain, and Behavior in Birds, MIT Press, Cambridge, MA, 1993, pp. 77 98. [48] Wang, Y-C., Jiang, S. and Frost, B.J., Visual processing in pigeon nucleus rotundus: Luminance, color, motion, and looming subdivisions, Vision Res., 10 (1993) 21 30. [49] Wilson, P., The organisation of the visual hyperstriatum in the domestic chick. II. Receptive field properties of single units, Brain Res., 188 (1980) 333-345. [50] Zappia J.V. and Rogers L.J., Light experience during development affects asymmetry of the forebrain function in chickens, Dev. Brain Res., 11 (1983) 93 106. [51] Zappia, J.V. and Rogers, L.J., Sex differences and the reversal of brain asymmetry by testosterone in chickens, Behav. Brain Res., 23 (1987) 261-267.
BEHAVIOURAL BRAIN RESEARCH Behavioural Brain Research 80 (1996) 65-73
Research report
Exposure to different wavelengths of light and the development of structural and functional asymmetries in the chicken L.J. R o g e r s *, G . A . K r e b s
Department of Physiology, University of New England, Armidale, N.S.W. 2351, Australia Received 11 July 1995; revised 16 January 1996; accepted 22 January 1996
Abstract The thalamofugal visual projections of the chick are known to develop in response to stimulation by light prior to hatching, and asymmetry in the number of projections develops as a consequence of the embryo being oriented in the egg so that it occludes its left eye. The right eye only is stimulated by light and this causes the visual projections connected to the right eye to develop in advance of those connected to the left. We have now found that exposure of embryos, from day 19 of incubation to hatching, to red (peak transmission at 670 nm) or green (peak at 500 nm) light is as effective as broad-spectrum (white) light in establishing asymmetry in these projections. The intensities of the light to which the embryos were exposed in each case were equivalent, achieved in part by removing the air sac end of the egg shell. The thalamofugal visual projections, therefore, develop in response to light stimulation but appear to have no wavelength specificity. This result is consistent with the apparent lack of involvement of the thalamofugal visual pathway in colour vision. However, functional asymmetry, tested as left-right eye differences in categorising grain from pebbles, was found to be less marked in the chicks that had been exposed to green light compared to those that had been exposed to 'white' light, and it was absent in those exposed to red light or incubated in the dark. Thus, there is wavelength specificity for the development of the behavioural asymmetry, which suggests involvement of colour-coded neurons outside the thalamofugal visual pathway, probably in the tectofugal pathway. Exposure of the embryos to red and green light alternating at 30 min intervals is as effective as 'white' light for establishing both the structural and functional asymmetry.
Keywords: Thalamofugal projections; Development; Wavelength specificity;Asymmetry;Visual behaviour
1. Introduction Birds have two main visual pathways, one which projects from the tectum to the ectostriatal region of the forebrain and the other which projects from the thalamus to the visual Wulst or hyperstriatum 1-18]; see Fig. 1. Structural asymmetries are present in both of these visual pathways [36]. Giinttlrk~in et al. [14] have shown that there is asymmetry in the tectofugal visual pathway of the pigeon. In layers 10-15 of the optic tectum the cells are larger and have more dendrites on the right side. A more recent study 1-22] has shown that the larger cell size on the right side is confined to layer 13 of the rectum and another study has shown that the cells in layers 2-7 of the tectum are larger on the left side [ 12]. *Corresponding author. Fax: +61-67-73-3234; E-mail: [email protected]. 0166-4328/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S016-64328 (96)00021-6
The cells in layers 2 - 7 receive input from the retina and they project to layer 13. The cells in layer 13 of the tectum receive input from the tectal commissures, the hyperstriatum and the archistriatum, and they project to the nucleus rotundus [6]. Their larger size on the right side may indicate more input from the contralateral tectum than their counterparts on the left side and also that they give rise to more projections to the rotundal nuclei. In fact, GOnt~irkOn and Melsbach [13] have reported that the left nucleus rotundus receives afferents from both sides of the optic tectum, with only a slightly greater bias for more from the ipsilateral tectum, whereas the right nucleus rotundus receives afferents almost exclusively from the right rectum. That is, inputs from the right eye are relayed firstly to the left tectum and from there almost exclusively to the left nucleus rotundus, whereas inputs from the left eye are relayed to the right tectum and from there to both rotundal nuclei. It is known that there are bilateral projections to the
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Fig. 1. The two visual pathways in the avian brain are illustrated in a simplifiedform. Note that the thalamofugal visual projections,arising from a collectionof nuclei in the thalamus known as the opticus principalis (OPT), feed forward to the hyperstriata on both sides of the forebrain.The contralateral projections are indicated by the grey lines. Note that input from the eye (grey lines) goes to the contralateral OPT and the contralateral tectum opticum (TO). The ipsilateral projections are indicated by the black lines. The stippled area in the hyperstriatum (HA and HD) indicates the approximate site of the dye injection and its spread from that site. Other abbreviations: Hp, hippocampus; HV, hyperstriatum ventrale; ROT, nucleus rotundus; E, ectostriatum;N, neostriatum; HA, hyperstriatum accessorium;HD, hyperstriatum dorsale.
rotundal nuclei in chicks [24], as in the pigeon, but so far there has been no investigation of asymmetry in these projections in chicks. Asymmetry in the thalamofugal visual pathway has been shown in the chicken [1,42]. The left side of the thalamus, which receives input from the right eye only, sends a larger number of projections to the hyperstriaturn than does the right side of the thalamus, which receives input from the left eye only. This asymmetry in the thalamofugal projections was first reported by Boxer and Stanford [5], who injected horseradish peroxidase into the hyperstriatum of 8-day-old chicks and located the asymmetry in the contralateral projections from either side of the thalamus to the hyperstriatum. This result has been confirmed by injection of retrograde labelling fluorescent dyes on day 2 or day 12 of posthatching life [1,42,44]. The above technique has revealed that the asymmetry is present in both males and females, but it is greater in males, at least on day 2 post-hatching [27]. The asymmetry of the visual projections is consistent with the lateralization of function present in young chicks [3,10,37]. For example, when tested monocularly, chicks using the right eye perform better in visual discrimination tasks [10,23,51]. Chicks using the left eye are more attentive to topographical cues and respond to small changes in the stimuli, whereas chicks using the right eye pay more attention to colour and respond only to large changes in the stimuli [2,28,34,45]. Although asymmetry on visual tasks has been reported to occur
in both males and females [23], in some studies females show a lesser degree of asymmetry than males [3,51]. The lateralization of the visual projections and of visual function in the chick has been shown to depend on exposure of the embryo to light during the last 3 to 4 days prior to hatching. During this stage of development, the embryo is oriented in the egg so that the right eye is positioned next to the air sac and the left eye is occluded by the body [31]. Thus, only the right eye receives stimulation by light entering through the shell and membranes. Chickens hatched from eggs that have been exposed to two or more hours of broad-spectrum, incandescent or fluorescent light on day 19/20 of incubation show lateralization for control of visual discrimination performance, whereas chickens hatched from eggs incubated in the dark show no asymmetry [32,40,50]. Furthermore, it is possible to reverse these asymmetries by withdrawing the embryo's head from the egg, occluding the right eye and allowing the left eye to receive stimulation by light [33]. The structural asymmetry in the visual projections is affected by stimulation by light in a similar manner. Exposure of the embryo's left eye to light reverses the direction of the asymmetry in the thalamofugal visual projections [42] and no asymmetry is present in chicks incubated in darkness [40]. Thus, stimulation by light prior to hatching influences the growth of the visual neurons which project from the thalamus to the forebrain [36]. The same dependence on light stimulation occurs for the development asymmetry in the tectofugal path-
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way: pigeons hatched from eggs incubated in darkness have no asymmetry of cell size [ 12]. It should be noted that the pigeon is an altricial species, hatching at an earlier state of development compared to the chicken. The earlier effect of light exposure on asymmetry in the tectofugal pathway may reflect its apparent earlier development compared to the thalamofugal visual pathway, as known to occur in chicks [38,39]. Another interesting comparison between the two visual pathways in birds is the involvement of the tectofugal pathway, but not the thalamofugal pathway, in colour vision [15,26,46]. Whereas the two pathways have different functional roles for colour vision, it is not known whether the same differentiation occurs for their light-stimulated development. Development of the tectofugal pathway might depend on wavelength, whereas development of the thalamofugal might not. We were, therefore, interested to see whether colour is a parameter of the light stimulation which generates the known structural and functional asymmetries in chicks, and we tested this by exposing embryos to different wavelengths of light.
67
One control group was exposed to the broad-spectrum incandescent light (without filtering) and another was incubated in darkness. (From here on, the groups will be referred to as green-light exposed, red-light exposed, alternating-light exposed, white-light exposed and darkincubated.) The term 'white-light' will be used to describe the broad-spectrum produced by the incandescent bulbs although it is recognised that they produce wavelengths largely over the red to green region of the spectrum and have relatively little output of the shorter wavelengths. The eggs were left undisturbed from day 19 of incubation until hatching. After hatching, the chicks were housed in groups of 2 to 3, and supplied food and water ad libitum. The home cages were fitted with clear perspex fronts (22 cm × 22 cm x 30 cm high). Warmth and light were provided by overhead 40 W incandescent bulbs (approximately 1000 lux). The cages were cleaned daily. The chicks were sexed either after they had been tested behaviourally or at the time of perfusion for histology. The behavioural data for males only is reported here, and histology was performed on the brains of males only. This choice was made because males are known to have a greater degree of lateralization than females [3,23,27,51].
2. Materials and methods
2.1. Animals and housing conditions White Leghorn × New Hampshire eggs were incubated in darkness until day 19 of incubation in a forceddraught, automatically turning incubator. On day 8 the eggs were candled using white light. On day 19, the eggs were divided into five equal groups. The egg shell was removed from the air sac end of the egg exposing the membranes over the head of the embryo. This procedure prevented the shell from impeding light input to the right eye and allowed precise control over the exposure to different wavelengths of light, as the wavelengths used might be filtered as they pass through the shell. After removal of the shell, the eggs were moved to Multiplo incubators which had been adapted for heating with an element rather than a light bulb. Light exposure of the eggs was achieved by placing incandescent lamps (40 W bulbs) over windows in the lid of the incubators. The intensity of light was adjusted using a rheostat so that the four groups of eggs were exposed to approximately 400 lux of light after filtering (measured using a Topcon Digital Illuminance Meter Model IM-3 placed alongside the eggs). One group was exposed to red light (incandescent 40 W bulbs filtered by a Rosco Supergel number 27 with transmission wavelength ranging from 620 to above 740 nm and a peak transmission at 670 nm; Fig. 2), another to green light (Rosco Supergel number 75 transmitting over the range of 460-580 nm with a peak at 500 nm; Fig. 2), and a third group was exposed to red and green light alternating at 30-min intervals.
2.2. Behavioural test for lateralization of brain function The ability to categorise grains of chick mash as distinct from pebbles was tested in a standard pebblefloor test requiring the chicks to search for grains of chick starter crumbles scattered on a background of pebbles [43]. The chicks were tested monocularly on day 8, following occlusion of one eye by a conicalshaped piece of adhesive tape applied 5 min before testing. The number of pecks at pebbles and grains were scored for a total of 60 pecks. Prior to testing, the chicks were deprived of food for 3 h. Only pecks involving new choices of grain or pebbles were scored. Error scores (pecks at pebbles) were recorded for each block of 20 pecks. The number of errors in the last block of 20 pecks (pecks 41-60) were the standard used to determine functional lateralization (for more detail see [34]). 2.3. Labelling the thalamofugal visual projections with fluorescent dyes
To determine the number of projections from each side of the thalamus to the forebrain, the retrogradely transported fluorescent dyes True Blue (Sigma T5891) and Fluorogold (Fluorochrome Inc.) were injected into the hyperstriatum, one into each side of the forebrain. The chicks to be injected with dye were taken from the same batches of eggs as were the chicks tested behaviourally, apart from the control groups incubated in darkness. The absence of asymmetry in chicks incubated in
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~
0.18
100
0.16
Wavelength (nm) Fig. 2. The percentage transmission spectra of the two filters is presented (dotted area for green filter and grey area for red filter, see text for details). The dashed line presents the absorbance spectrum for rod cells in the chick retina, as reported by Bowmaker and Knowles I-4]. The black arrow heads along the horizontal axis represent the positions of maximal absorption of the various cone cell pigments 1-47]. The white arrow heads indicate shifts that are calculated to occur due to the presence of red and yellow oil droplets [47J.
darkness and raised in identical conditions to those mentioned above has been reported previously [40]. The method of dye injection has been described previously in detail [ 1,40]. Briefly, on day 2 post-hatching, the chicks were anaesthetized with an intramuscular injection of 50 mg/kg ketamine and 5 mg/kg xylazine and placed in a stereotaxic apparatus. Using a Hamilton syringe, 0.6 #1 of a 5% solution of True Blue was then injected into the hyperstriatum aceessorium region of one of the hemispheres and 0.4 pl of a 4% solution of Fluorogold was injected into the same region in the other hemisphere. These different doses were used with the aim of obtaining approximately equal numbers of labelled neurons with each dye. The side into which each dye was injected was randomly assigned to control for any effects particular to each dye. After recovery, the chicks were returned to their home cages until they were 6 days old, when they were deeply anaesthetized with sodium pentobarbital and perfused with a solution of 10% formalin in 0.1 M phosphate buffer. The chicks were sexed by inspection of the gonads and the brains of the males were removed and sectioned into 40-/~mthick slices using a freezing microtome, with alternate sections mounted immediately onto gelatine-subbed slides. The sections were then dried and the slides were coverslipped. Specific regions in the forebrain and thalamus were identified with the aid of a stereotaxic atlas [19]. Inspection of the injection site in the hyperstriatum and of labelled cells in the thalamus was performed using ultraviolet microscopy. Injection site parameters for each dye were determined by examining sections of the fore-
brain. The distance from the rostral aspect of the brain to the section containing the greatest area of dye around the injection site, presumed to be the centre of the injection site, was determined. With the aid of an ocular micrometer grid, the distances from the centre of fluorescence to the midline of the brain and to the dorsal surface of the brain were measured on this section. The diameter of the area of fluorescence in this section was also measured to estimate the size of the injection site. Previous studies have shown that the region of spread of the dye around the injection site approximates a sphere [40,41 ], and hence the volume of this region was calculated from the measured diameter. From thalamic sections of each brain, the number of labelled cells ipsilateral and eontralateral to the injection site were determined, using the previously described method [40,41]. The total number of cells in the side of the thalamus contralateral (C) to the injection site was divided by the total number of labelled cells in the side of the thalamus ipsilateral (I) to the injection site to determine the C/I ratio for each dye/hemisphere. Use of this ratio controls for variations in the amount of dye injected [ 1].
2.4. Analysis of data The behavioural data was analysed using a generalised linear model with binomial error function and LOGIT LINK FUNCTION. Injection sites and C/I ratios were compared using two-factor ANOVAs and Pearson correlation. Post-hoe t-tests were applied, one-tailed only when the result could be predicted from previous studies.
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3. Results 3.1. Behavioural test In the pebble-floor test, there were significant main effects of light treatment (F4,84 = 3.10, 0.01 < P < 0.025) and of the eye used during testing (F1,84=70.93, P<0.001); see Fig. 3A. There was also a significant interaction between the light treatment and the eye used (F4,84 = 4.81, 0.001 < P < 0.005). As expected from previous studies, chicks exposed to white-light and tested using the right eye made significantly fewer pecks at pebbles in the last 20 pecks than those using the left eye (ts4=6.46, P<0.001). This was also the case for chicks that were exposed to green light, and to alternating red and green light (t84=3.79, P<0.002; and t84=4.56, P<0.002, respectively). However, in chicks
A ~
-v
16
14
69
exposed to red light there was no significant difference in performance tested using the left or right eye (ts4= 1.93, 0 . 1 0 < P < 0 . 2 0 ) , and the same was true in the dark-incubated chicks (ts4 = 1.53, 0.20 < P < 0.40). The degree of lateralization was greatest in white-light exposed chicks. Chicks that were exposed to green light during incubation and were tested using the right eye made significantly more pecks at pebbles than whitelight exposed chicks using the right eye (ts4=3.47, P<0.002). This also applies to chicks that were redlight exposed when they were tested using the right eye (t84=2.90, 0.002
"T"
3.2. Labelled visual projections
B
iil
*L
Y__
1 DARK
WH1TE
RED
GREEN
FLASH R/G
Fig. 3. Lateralization in behaviour and in the structure of the thalamofugal projections. A: The number of pecks at grains (as opposed to pebbles) in the last 20 pecks of the pebble-floortest in chicks tested monocularly using the left eye (black bars) or fight eye (white bars). B: C/I ratios (see text) followinginjection of tracer dyes into the left byperstriatum (black bars) or right hyperstriatum (white bars). Means and S.E.M.s are presented and asterisks represent significant left-right differences (P<0.05). The data are for chicks exposed to the various light conditions prior to hatching. Note that data for C/I ratios followingdark incubation (grey and dotted bars) are from a previous study [40].
Examination of the injection sites in the forebrain confirmed that the centres of all of the injection sites were located in the hyperstriatum accessorium (HA) and that injected dye had diffused into the region of the hyperstriatum dorsale (HD) and the nucleus intercalatus of the HA (IHA), the regions in which thalamofugal projections terminate [18]. The site of the dye injection was at a mean depth___S.E.M, of 1.38+_0.06mm from the dorsal surface of the brain. As in earlier studies [ 1,40], for both Fluorogold and True Blue, no significant correlation was found between the volume of the injection site in the forebrain and the C/I ratio ( Pearson correlation, r=0.13). The mean volume of the injection site was 1.03 +0.17 mm 3 for Fluorogold and 0.81 +0.18 mm a for True Blue. Analyses of variance indicated that the values for depth of injection and volume of the dye injected did not differ significantly among the four treatment groups or between the left and right hemispheres. ANOVAs of the data for the rostral displacement (1.11 ___0.04 mm) and lateral displacement ( 1.05 _+0.06 mm) did not show a main effect of left/right side (F3,42=0.006, P = 0 . 9 4 and F3,42=0.0003, P=0.99, respectively). However, the rostral and lateral displacements of the injection sites were found to differ significantly between the light treatments (F3.~2=3.00, P = 0.04, and F3,42=4.28, P - 0 . 0 1 , respectively), although on both sides injection sites were confined to the HA. This unintentional error in the placement of injections does not appear to have affected the values of the C/1 ratios, since no significant correlation was found between
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the C/I ratios and either the distance of the injection site from the midline (r= 0.3) or the rostral displacement
4. Discussion
(r = 0.09).
Asymmetry in the organisation of the thalamofugal visual projections of the young chick is known to develop following exposure of the embryo to broad-spectrum light [ 32, 36, 50] and the present results confirm this fact. Previous research indicates that the asymmetry is present in the contralateral projections [5,40] and, thus, there are more projections from the left thalamus to right hyperstriatum than from the right thalamus to left hyperstriatum. Here we have shown that stimulation of the right eye of the embryo by red or green light of equivalent intensity to the 'white' (or broad-spectrum) light generates the same asymmetry in 2-day-old chicks. This result should be compared to the previously reported absence of asymmetry in the thalamofugal visual projections of chicks hatched from eggs incubated in darkness [40] (see also Fig. 3B). The apparent lack of wavelength specificity for light-dependent development of the thalamofugal visual projections is consistent with the evidence that lesions of the hyperstriatum have little effect on colour vision [26] compared to lesions placed in the tectofugal visual pathway [15,25]. The role of the tectofugal pathway in colour vision is supported by the fact that cells in the dorsal-anterior region of the rotundal nuclei of the tectofugal pathway respond vigorously to specific colours [48]. We recognise that the experiments reported here need to be repeated with exposure of the embryo to other wavelengths of light before a firm conclusion can be reached, but it appears that the asymmetrical development and lateralized function of the thalamofugal visual pathway lacks wavelength specificity. The fact that exposure to alternating red and green light generates no greater asymmetry than exposure to either the red or green light alone indicates that both colours of light may influence the development of the same population of neurons. That is, the lack of an additive effect of the alternating red and green light indicates that there may not be separate populations of neurons that develop in response to stimulation by either red or green light. Indeed, there was an indication that the chicks exposed to the alternating red and green light might have a lesser degree of asymmetry than any of the other groups but this was not significant. Of course, it might be argued that these chicks received insufficient exposure to either colour of light to promote the development of asymmetry, but the total amount of exposure to each colour exceeded 24 h, a sufficient period of exposure for broadspectrum light to cause structural asymmetry [42]. We also investigated the effects of exposing the embryo to the different colours of light on the lateralization of visually guided behaviour tested on day 8 post-hatching. It is known that exposure of the embryo to white light on day 19 of incubation for as little as 2h leads to lateralization of performance on the task requiring the
Labelled cells were confined to the dorsolateral anterior thalamus (DLA) (rostrolateral and lateral parts), in the region of the nucleus opticus principalis thalami (nOPT), a region in the thalamofugal pathway which receives direct input from the retinae and projects to the HD and IHA [7,181. Photographs and detailed diagrammatic representations of both the projection sites in the forebrain and the labelled cells in the thalamus have been published previously [ 1,42]. The C/I ratios were analysed by a 2-way ANOVA (light xside). No main effect of light was found (F3,42 = 1.31, P=0.28), but there was a significant main effect of the side injected (F1.42= 14.98, P=0.0004). There was no significant interaction ( F 3 , 4 2 = 0 . 3 4 , P = 0.80). Thus, all of the light conditions produced asymmetry (see Fig. 3B). Since the two hemispheres of each brain were injected with different dyes, left and right C/I ratios of individuals could be compared using paired statistical tests. In the group of chicks exposed to white-light, the mean C/I ratio following injection of the right hemisphere was significantly higher than that following injection of the left hemisphere (Fig. 3B; one-tailed paired t-test, P = 0.001). This asymmetry is in agreement with previous studies of untreated, light-incubated male chicks [1,40,42]. Structural asymmetry was found also in the chicks exposed to red, green and alternating red and green light (paired t-tests, P=0.018, P=0.02, and P = 0.04, respectively). Comparison of the left-right differences in the C/I ratio calculated as ( R - L / R + L) values revealed that the same degree of asymmetry was present in all groups (F3.22 = 1.1, P=0.37; Fig. 4).
0,4-
0.3-
02-
lil l iii!i
0.1-
WHITE
RED
iiiiiiii !ill GREEN FLASHR/G
Fig. 4. The C/I ratios for injections into the left and right sides of the hyperstriatum have been expressed as ( R - L / R +L) for each individual. This figure presents the mean values for each group with S.E.M.s. There is no significant difference between any of the groups, although there is a trend for less asymmetry to be present in the thalamofugal visual system of the group exposed to alternating red and green light (Flash R/G).
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chick to shift from pecking randomly at pebbles and grain to pecking at grain only [33]. This lateralization has been shown both by unilateral treatment of the forebrain hemispheres with either cycloheximide or glutamate and by monocular testing [30,35]. Drug treatment of the left, but not the right, hemisphere impairs performance on this task [17,37] and occlusion of the right, but not the left, eye produces the same impairment [23,51]. Thus, the task is best performed when the chick uses the right eye and the left hemisphere. The results reported here show that this advantage of the right eye and the left hemisphere does not develop if the embryos are incubated in darkness. Although following incubation in the dark there was a slight tendency for the group of chicks using the right eye to perform better than the group using the left eye, this difference was not significant. In fact, in the chicks incubated in darkness, use of neither the left or right eye allowed the chick to shift away from pecking randomly at pebbles and grain. This result correlates with an absence of asymmetry in the thalamofugal visual projections of chicks incubated in darkness [40]. Exposure of the embryos to red light failed to establish lateralization of performance on the pebble-floor task, whereas exposure to green light did produce significant lateralization. Therefore, the development of this particular functional asymmetry is wavelength dependent. Red light was clearly less effective than green light. The chicken has cone pigments which give rise to three peaks of maximal absorption between 400-500 nm, another at about 500 nm and yet another around 570 nm [4,21,46,47]; see Fig. 2. The rods have an absorbance maximum at 506 nm and a cut off around 600 nm in the longer wavelength direction [4]; see Fig. 2. Thus, the red filter that we used would stimulate cones and not rods, whereas the green filter would stimulate both rods and cones. In fact, the transmission range of the green filter overlaps almost exactly the absorbance spectrum of the rods. The lesser effect of exposure to red light, compared to green, in establishing the behavioural asymmetry might possibly depend on a lack of stimulation of rod cells in the retina although the wavelength specificity might be explained equally well by selective stimulation of the different populations of cone cells. Judging from results obtained with pigeons, the behavioural ability to discriminate between different wavelengths is better for the shorter wavelengths, which includes the green region of spectrum, than for the longer wavelengths above 600 nm [46]. Better ability to discriminate in the green region of the spectrum is also consistent with the known absorbance spectrum for the retinal photoreceptors and oil droplets [4]. Therefore, the wavelengths that we have found to be most effective for stimulating development of the visual pathways can also be discriminated best by the bird. It should, however, be noted that Wang et al. [48] have found in
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pigeons that most of the colour-coded cells in the dorsalanterior part of the nucleus rotundus are more sensitive to wavelengths in the red and the blue regions of the spectrum, with low responsiveness to green. This apparent contradiction cannot be reconciled with the behavioural studies, nor with our results. Our finding that exposure of the embryo to red light leads to asymmetry in the thalamofugal visual projections but not lateralization of performance on the pebblefloor task suggests the thalamofugal visual pathway may have either no role or a minor role relative to the tectofugal visual pathway in this particular visually guided behaviour. Previously, one of the authors [30] suggested that performance on the pebble-floor task relies primarily on use of the thalamofugal pathway, based on the age-dependent loss of functional asymmetry in parallel with the loss of asymmetry in the thalamofugal pathway by day 21 post-hatching. This conclusion would now seem to be incorrect. Moreover, the binocular frontal field of vision is used for pecking in the pebblefloor task and it is known to be exclusively tectofugal in the pigeon [14]. As a chick searching for grain on the pebble-floor task uses both the frontal and lateral visual fields, it is likely that both visual pathways are used in performing this task. There is also evidence for communication between the two visual pathways via projections from HA first to the hyperstriatum ventrale and then to the peri-ectostriatal belt and also between HA and the optic tectum [8,9]. The function of the thalamofugal visual pathway remains unclear. The tectofugal visual pathway is known to be involved in colour vision, visual acuity, detection of movement and pattern discrimination [11,29,48], whereas the thalamofugal visual pathway may be involved in pattern discrimination and detection of movement, but not in acuity or, as mentioned above, in colour vision [14,20,49]. There is some evidence that the processing of thalamofugal inputs to the forebrain enhances performance on visual discrimination tasks, but that the tectofugal inputs provide the essential processing [16]. In the task used in this study, the grains cannot be distinguished from pebbles on the basis of colour, size or shape. They can be distinguished by differences in texture and brightness and, because the grain can be moved by either scratching or pecking with a closed beak, they might also be distinguished by movement relative to the background. Both visual pathways might contribute to performance on this task, but it is likely that the acuity abilities of the tectofugal pathway play an essential part in guiding the shift to pecking almost exclusively at grain. If so, our results suggest that asymmetry might be established in the tectofugal pathway by exposing the embryo to green, but not red, light. This would explain the dissociation between asymmetry in the thalamofugal pathway, caused by red light, and the lateralized performance of the
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pebble-floor task. Moreover, wavelength specificity for the development of the tectofugal pathway would be consistent with the clear colour specificity of neurons within the nucleus rotundus [48]. Alternatively, the asymmetry generated by exposing the embryo to green, but not red, light and manifested in the behavioural task may occur at a higher level of cognitive integration, outside the primary visual pathways. Exposure of the embryos to alternating red and green light also produced significant lateralization of performance on the pebble-floor task. Unless red light can synergise with green light, even though it has no effect on its own, the lateralization must be caused by the exposure to green light for half of the entire period used in this experiments (i.e. for about 24 h). It would now be interesting to know whether, as for broad-spectrum light, as little as 2 h of green light is sufficient to generate the lateralization. These results show clear effects of light of different wavelengths on the development of functional and structural asymmetries in the chick. It appears that exposure of the embryo to green light may generate asymmetry in both of the visual pathways, whereas exposure to red light may do so only in the thalamofugal visual pathway. In this study we have tried to establish associations between asymmetry in a neural pathway and behaviour, and to draw links between the effects of sensory stimulation on the development and the functional specificity of these neurons. The thalamofugal visual projections appear to lack wavelength specificity both for stimulation of their development and for their later use in visual perception.
Acknowledgement This research was supported by an ARC grant number A09130918 to L.J. Rogers. We thank to Dr. A.N.B. Johnston for helpful suggestions.
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