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Chemosensory input and lateralization of brain function in the domestic chick
Behavioural Brain Research 133 (2002) 293 /300 www.elsevier.com/locate/bbr
Research report
Chemosensory input and lateralization of brain function in the domestic chick Thomas H.J. Burne *, Lesley J. Rogers Centre of Neuroscience and Animal Behaviour, School of Biological, Biomedical and Molecular Sciences, The University of New England, Armidale 2351, Australia Received 15 October 2001; received in revised form 2 January 2002; accepted 2 January 2002
Abstract One-day old domestic chicks (Gallus gallus domesticus ) show concentration-dependent behavioural responses to olfactory cues. In the present study we investigated the lateralized olfactory responses of 1-day-old chicks to the odours of eugenol and iso -amyl acetate. In experiment 1 different concentrations of each odour were presented in repeated trials to chicks housed individually. The odours were presented together with a small coloured bead at which the chick pecked. When tested with the highest concentration of eugenol (100% v/v), the chicks demonstrated more head shaking when their left nostril was occluded (RN; right nostril in use) than when their right nostril was occluded (LN; left nostril in use). No such lateralization occurred in response to iso -amyl acetate. This result was confirmed in a second experiment in which the chicks were tested with unscented stimuli, 100% eugenol and 100% iso amyl acetate. In experiment 3 we found that occluding both the chicks’ nostrils abolished the head shaking response to eugenol and to iso amyl acetate. Thus, the chicks’ head shaking responses to the odorants eugenol and iso -amyl acetate are mediated primarily by inputs from within the nasal cavity, and not by oral or occular inputs. The present results are consistent with the hypothesis that there is lateralization to olfactory cues and that it is dependent on the involvement of receptors inside the nasal cavity. We suggest that differences in lateralized olfactory responses to different odours are affected by the relative involvement of intranasal olfactory and trigeminal chemoreceptors. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Domestic chicks; Chemosensory input; Lateralized olfactory responses; Olfaction
1. Introduction Differential use of the forebrain hemispheres by the chick has been established largely in response to visual cues [2 /4,16,24]. Studies in which chicks have been imprinted on an object or presented with a small bead and tested monocularly show specialized roles of the left and right hemispheres. It has been shown that the left hemisphere assigns stimuli to categories for considered responses, whereas the right hemisphere controls more immediate responses and attends to spatial cues [2,3,5,24].
* Corresponding author. Present address: Laboratory of Cognitive and Developmental Neuroscience, The Babraham Institute, Cambridge CB2 4AT, UK. Tel.: 44-1223-496-435; fax: 44-1223496-028 E-mail address: [email protected] (T.H.J. Burne).
The domestic chick uses odours in the formation of attachments (see [14] for review) and there is evidence that the hemispheres may be lateralized for controlling responses to a familiar odour. Vallortigara and Andrew [25] exposed chicks to clove oil odour contained in a small cylinder suspended in the home cage. On day 3 posthatching, each chick was tested in a runway with a cylinder containing the familiar odour suspended at one end and a similar cylinder not scented with clove oil at the other end. The chicks were tested with either the left or right nostril occluded with wax. Those with the left nostril occluded (right nostril in use; RN) chose to approach the clove oil scented stimulus in preference to the unscented stimulus. Chicks with the right nostril occluded (left nostril in use; LN) made no choice between the two stimuli. In another experiment Vallortigara and Andrew [25] reared chicks with an unscented cylinder and tested them in a laneway with a scented cylinder at one end (1
0166-4328/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 2 ) 0 0 0 0 9 - 8
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ml of n -amyl acetate, 1 ml of amyl acetate or 1 ml of orange oil) and an unscented cylinder at the other end to examine the chicks’ response to a novel odour. They found that RN chicks approached the familiar-unscented cylinder (so avoiding the novel-scented cylinder), whereas LN chicks approached both stimuli at random. Thus, RN (but not LN) chicks show a lateralized avoidance response to a novel odour, as well as a lateralized approach response to a familiar odour. Vallortigara and Andrew [25] also presented clove oil to chicks within a small peckable target, which was a grey, metal box containing a clean piece of cotton soaked with five drops of clove oil. The odour could diffuse out of the box through small holes in its walls. At test, 3-day-old male chicks, which had been reared with an unscented table tennis ball suspended in their cage, were presented with odour in four consecutive 10 s trials with either the left or right nostril occluded. A greater percentage of RN chicks (/70%) compared to LN chicks shook their heads on the first trial, indicating that they had detected the clove oil. Clove oil is believed to be a pure odorant that stimulates olfactory receptors in the nasal epithelium only and not the taste receptors or the free nerve endings of the trigeminal nerve in the oral cavity [9]. Occlusion of a nostril confines odour stimulation to the epithelium of the unoccluded nostril as the nasal cavities of chicks are separated by a cartilaginous septum [15]. The olfactory epithelium in each nasal cavity projects, via the olfactory nerve, to its ipsilateral olfactory bulb only [10], whereas the trigeminal inputs from each nasal cavity go to both hemispheres. Hence, unilateral stimulation of one nostril would lead to processing trigeminal inputs by both forebrain hemispheres due to stimulation of trigeminal receptors, but stimulation of olfactory receptors would involve primarily the ipsilateral forebrain hemisphere. Lateralized responses to odorants might be more likely to occur within the olfactory system, with its predominantly ipsilateral connections, rather than the trigeminal system. Thus, the finding that a preference for the familiar odour is expressed by chicks using the right but not the left nostril could be interpreted as indicating that the right hemisphere alone is specialized to process olfactory inputs and/or to control responses to odours. However, ipsilateral connections do not, in themselves, imply lateralization, unless the hemispheres which receive these projections process that information differentially. We have developed a behavioural task in which newly hatched chicks are allowed to peck at a small bead in the presence of an odorant [7]. The number of pecks directed at the bead and the amount of head shaking (a rapid, lateral movement of the head) that occurs during brief (10 s) trials is measured. Using this task we have shown that 1-day-old chicks demonstrate increased
amounts of head shaking and decreased amounts of pecking to increasing concentrations of some odorants, such as iso -amyl acetate and eugenol (eugenol is the primary component of clove oil and eugenol smells like clove oil to humans). We chose to use iso -amyl acetate in this experiment because it has been used widely in experiments in both mammals [14,21] and birds [26], and we have previously looked in detail at chicks responses to this odorant using this task [7]. All the above olfactory tasks [7,25] required the chick to process visual and olfactory input and thus the lateralization may reflect hemispheric specialization for the integration of visual and olfactory information. Rogers et al. [17] showed that head shaking to clove oil occurs for RN only when the bead is blue and not when it is red. Thus, there is an interaction between visual and olfactory cues. It appears that the visual cues override olfactory cues in the left hemisphere (LN condition) and that the left hemisphere ignores olfactory cues when the visual cues are very attractive, as in the case of the blue bead. The main question addressed in the experiments reported here is whether the lateralization of head shaking response occurs with eugenol and iso -amyl acetate, and over a range of doses. We predicted (based on previous findings [25]) that chicks using their left nostril (LN) would not shake their head, whereas RN chicks would, following the presentation of a range of concentrations of eugenol or iso -amyl acetate. To confirm the results of Experiment 1 we conducted a second experiment in which we presented chicks tested either using the LN or RN with either unscented stimuli or a single concentration of either eugenol or iso -amyl acetate. To rule out the involvement of receptors outside the nasal cavity in the response to eugenol and iso -amyl acetate we conducted a third experiment in which chicks were tested with both nostrils occluded.
2. Methods 2.1. Animals and housing Seventy-two chicks were used in this study, a separate group of 24 chicks being used in each of the three experiments. Fertile White Leghorn /Australorp eggs (Barter and Sons, Huntingwood, Australia) were incubated for the first 17 days in a forced-draft, turning incubator (Multiquip, Austral, Australia). The incubator was maintained at 37 /38 8C and 80% relative humidity. Viable eggs were transferred to a hatching incubator maintained at 37 /38 8C and 95% relative humidity from day 17 of incubation until 18 h posthatching. An illuminated heat lamp provided heat and light (100 /200 lx, depending on the position of the egg on the hatching tray). Eighteen hours after hatching
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each chick was housed individually in a clean grey metal cage (21 /21 /31 cm) on a layer of paper toweling and situated on a three-tier rack. The home cages consisted of galvanized steel on three sides and transparent plastic on the other. Warmth and illumination were provided by 40-W pearlescent bulbs and the temperature within the home cage was maintained at 29 /33 8C. Feeding and drinking were encouraged by sprinkling 5 g of chick starter mash (Fielders, Tamworth, Australia) in front of the chick and by dipping its beak into a water drinker located at the rear of the home cage. Thereafter the chicks remained undisturbed for 2 /6 h before they were tested. 2.2. Testing stimuli The test stimulus used has been described previously [7,8]. Briefly, it consisted of a coloured plastic bead (4 mm diameter) affixed to a white opaque 500 ml COBAS plastic sample cup which was attached to the end of a 250 mm long glass rod (4 mm outside diameter). The chicks were presented with the odour of eugenol or iso amyl acetate by applying 10 ml of the odorant to cotton wool contained in the sample cup. Different concentrations of odorant were prepared by making serial dilutions in ethanol and applying 10 ml of each dilution of solution to the cotton wool. We used the following dilutions; 0, 0.01, 0.1, 1, 10 or 100% (v/v). The walls of the sample cup were perforated with 15 evenly spaced 0.5 mm diameter holes to allow for adequate dispersal of odorant. We chose to present a range of bead colours, including yellow, light green, dark green, light blue, dark blue and red. This method was used to avoid habituation of pecking, which occurs rapidly in the chick [1], over repeated trials. A white bead was used during the training trials only. 2.3. Testing conditions Chicks were tested in a separate grey metal cage, referred to as the testing cage, that had the same dimensions as the home cage. Their behaviour was recorded on video tape by a centrally situated video camera located directly above the testing cage and connected to an external monitor. The front of the cage consisted of a metal sheet with 9 holes (7 mm diameter) spaced evenly in two rows approximately 50 and 70 mm from the floor of the cage. The testing room was visible through these holes and they served to direct the chick’s attention towards the front of the cage. The testing stimulus was introduced into the testing cage through a tenth, centrally placed hole (18 mm diameter). The testing cage was illuminated with a single 40-W pearlescent bulb. To prevent the chick from seeing the experimenter, the image from the monitor was used to guide the bead to a central position 5 mm from the tip of
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the chick’s beak. Although this testing procedure meant that the chicks received extra handling (they were carried gently ca. 1.5 m from the home cage to the testing cage), it enabled standardization of the test environment. 2.4. Occluding nostrils The chicks had one or other nostril occluded temporarily with an unscented wax preparation (Tender Beauty Products, Newcastle, Australia) 10 min before testing. The wax preparation was relatively easy to apply and, when applied to the chick’s beak, remained in place for at least 24 h. The wax was softened by placing it into a glass vial immersed in warm water. It was then applied to one or other of the chick’s nostrils using a small metal spatula such that it completely covered the external opening and a small portion of the surrounding beak. The application of a thin layer of the wax (less than 2 mm) to the nostrils did not obscure the chick’s vision. Some of the chick’s initially attempted to remove the wax plug by wiping their bill on the floor of the cage and scratching at their bill with their feet. However, none of the wax plugs became dislodged. In experiment 1 either the chick’s left (RN: right nostril in use) or right nostril (LN: left nostril in use) was occluded. In experiment 2 half of the chicks had their left nostril occluded in the first testing trial and their right nostril occluded in the second testing trial, and the remainder had the reverse condition (right nostril occluded in the first trial and left nostril occluded in the second trial). In experiment 3 half of the chicks had both nostrils occluded (Oc), and the remainder had unobstructed nostrils (BN: both nostrils in use). 2.5. Testing procedure Testing began when each chick was 20 /24 h posthatching and involved presentation of a white bead attached to an unscented sample cup for two 20-s trials in the testing cage. These ‘training trials’ were used to familiarize the chicks with the testing apparatus. In Experiment 1 four separate groups of six chicks were tested repeatedly over six trials with 0, 0.01, 0.1, 1, 10 or 100% (v/v) concentrations (presented in a Latin-square design) of one or other of the odorants (eugenol or iso amyl acetate). In Experiment 2 three separate groups of eight chicks were repeatedly tested over two trials with either unscented stimuli or 100% (v/v) concentrations of one of the odorants (eugenol or iso -amyl acetate). In Experiment 3 four separate groups of six chicks were repeatedly tested over four trials with 0, 1, 10 or 100% (v/v) concentrations of one of the odorants (eugenol or iso -amyl acetate). In Experiments 1 and 3 the colour of bead used in each test was allocated in a Latin-square design. The stimuli were presented to the chick in 10-s
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trials separated by 10-min intervals during which the chick was returned to its home cage. In Experiment 2 a chrome, coloured bead was used in both trials and the inter-trial interval was increased to 20 min so that the wax occluding one of the chicks’ nostrils could be removed and the contralateral nostril occluded. The handling procedure may have altered the chicks’ level of responding, although it was noted that the chicks were not unduly stressed and were, in the main, motivated to peck at the bead during each trial. Also, any effects of handling were consistent between treatments. In all of the trials the number of pecks at the bead and the number of bouts of head shaking were recorded during each of the 20 s (training) and 10 s (testing) periods using video tapes. The number of pecks directed at the glass tube or sample cup was not scored; more than 95% of pecks were directed at the bead. A bout of head shaking was scored when at least two rapid alternating lateral movements of the head occurred within 0.1 s. A separate bout of head shaking was scored after the head remained stationary for 0.4 s. Although lateralized responses for head shaking and not pecking have been reported previously [17,25] both data sets for pecking and head shaking are presented.
3. Results
main effect of the factor ‘Nostril’ (F1,20 /0.5, P /0.5) or ‘Colour’ (F5,100 /0.8, P /0.6) on head shaking and the interaction of the main effects of Odour, Nostril and Dose failed to reach significance (F5,100 /1.7, P /0.15). Inspection of the data presented in Fig. 1 suggests that any effects of Nostril on head shaking were restricted to the highest concentration of eugenol presented. Analysis of the head shaking responses to 100% concentrations revealed a significant main effect of Odour (F1,20 /7.4, P /0.01) and a signifcant interaction of Odour and Nostril (F1,20 /5.6, P / 0.03). There was a significant difference between the head shaking response of LN and RN chicks to the highest concentration of eugenol presented (100% v/v; t10 /3.1, P /0.01; Fig. 1a). The number of bouts of head shaking by RN chicks presented with 100% eugenol was also significantly higher than that given to the control stimulus (0%; t10 /3.1, P /0.01). This contrasts with the responses of LN chicks, which had similar head shaking scores for all concentrations of eugenol presented. There were no significant differences between the head shaking responses of LN and RN chicks to any concentration of iso -amyl acetate presented (Fig. 1b), even though concentration /response curves were evident for chicks tested with LN and RN. There was no significant effect of Colour on the head shaking data (F5,100 /0.8, P / 0.6). There was a non-significant tendency for an effect of the main factor Colour on the pecking scores (F5,100 / 2.2, P /0.06). Overall chicks pecked highest at light blue coloured beads (6.39/0.7) and least at yellow coloured beads (3.49/0.7), with an intermediate number of pecks given to the remaining differently coloured beads. There were no significant effects of the other main factors or their interactions on the pecking scores (P / 0.10). The proportion of chicks responding at each dose of each odorant is shown in Table 1. The number of chicks pecking did not vary in response to the presentation of each concentration of odorant. By contrast the number of LN and RN chicks head shaking was highest in response to the two highest concentrations of iso -amyl acetate presented (10 and 100%). The number of RN chicks head shaking was highest at the highest concentration of eugenol presented (100%), whereas the number of LN chicks head shaking did not increase in response to the highest concentration of eugenol presented (100%).
3.1. Experiment 1
3.2. Experiment 2
There was a significant main effect of the factor ‘Dose’ on the chicks’ head shaking responses (F5,100 / 14.1, P B/ 0.001). There was also a significant effect of the interaction between the main factors ‘Odour’ and Dose (F5,100 /5.2, P B/ 0.001). There was no significant
There was a significant effect of Odour on the chicks head shaking (F2,21 /9.7, P B/0.01) and pecking responses (F2,21 /4.8, P /0.02). As can be seen in Fig. 2 the chicks had high pecking and low head shaking scores to unscented stimuli and low pecking and high head
2.6. Statistical analysis The data were square-root transformed (n/1) and analysed using repeated measures ANOVA or one-way ANOVA. In Experiment 1 the main factors analysed were Odour (eugenol or iso -amyl acetate), Nostril (LN or RN) and the repeated measures were fitted on the factors Dose (0. 0.01, 0.1, 1, 10 or 100% concentrations) and Colour (red, yellow, light blue, dark blue, light green and dark green). In Experiment 2 the main factor analysed was Odour (unscented, eugenol or iso -amyl acetate) and the repeated measure was fitted on the factor Nostril (LN or RN). In Experiment 3 the main factors analysed were Odour (Eugenol or iso -amyl acetate) and Nostril (occluded or unobstructed), and the repeated measure was fitted on the factor Dose (0, 1, 10 and 100). Post-hoc analysis was performed with either paired or unpaired t -tests. Statistical values with a probability value (P ) of B/0.05 were considered to indicate significant differences between groups.
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Fig. 1. Mean (9S.E.M.) head shaking (a, b) and pecking (c, d) responses of chicks tested with various concentrations of (a, c) eugenol or (b, d) iso amyl acetate. Four separate groups of chicks (n 6 per group) had either the left (right nostril in use, RN, m) or right nostril (left nostril in use, LN, k) occluded during testing. They were tested repeatedly and presented with stimuli (bead colour and odorant concentration) in a Latin-square design. *P B 0.05, paired t -test.
shaking scores to iso -amyl acetate. The chicks had intermediary head shaking and pecking responses to eugenol. There was a significant interaction of the factors Nostril and Odour (F2,21 /8.2, P /0.002) on the head shaking scores but not on the pecking scores (F2,21 /0.4, P /0.7). Post-hoc analysis with paired ttests indicated that chicks had higher head shaking scores when they were using their RN, rather than their LN, in response to eugenol (t7 /5.8, P B/0.01) but not to iso -amyl acetate (t7 /0.3, P /0.8) or unscented stimuli (t7 /0.6, P /0.6). This confirms the results found in Experiment 1. 3.3. Experiment 3 There was a significant main effect of Nostril (F1,20 / 10.4, P /0.004) and Odour (F1,20 /5.8, P /0.03) on the head shaking response. The interaction between Nostril (BN or Oc) and Odour failed to reach significance (F1,20 /3.5, P /0.08). There was a significant main effect of Dose on the head shaking scores (F3,60 /7.0, P B/0.001). It can be seen in Fig. 3 that there appeared to be a trend for the head shaking scores of Oc chicks to increase with increasing concentrations of iso -amyl acetate and eugenol. Chicks tested with both nostrils in use (BN) had significantly higher head shaking scores than chicks with both nostrils occluded (Oc) at the 100% concentration for iso -amyl acetate (t10 /4.0, P B/0.01). There was a tendency for BN chicks to shake their head more than Oc chicks to 10% iso -amyl acetate (t10 /1.9, P /0.09) and 100% eugenol (t10 /2.1, P /0.06).
There was a significant main effect of Nostril on the pecking response (F1,20 /8.5, P /0.009); Oc chicks had higher pecking scores, overall, than BN chicks. BN chicks pecked significantly less than Oc chicks to 10% (t10 /3.5, P /0.005) and 100% iso -amyl acetate (t10 / 5.8, P B/0.001). There were no significant effects of the other main factors (Dose or Odour) or their interactions on the pecking scores.
4. Discussion The main result of this study was that there was a lateralization in head shaking in response to the highest concentration of eugenol but not of iso -amyl acetate. This result was confirmed in a second experiment in which only the highest concentration of eugenol and iso amyl acetate were presented. The results from a third experiment, in which the chicks were tested with both nostrils occluded, ruled out the involvement of extra nasal receptors, such as those from oral or occular inputs in association with the odours tested. Therefore, 1-day-old chicks do not show a right nostril advantage for the perception of all odorants. One possible explanation for the present findings is the relative involvement of the olfactory and trigeminal receptors within the nasal cavity. The principle olfactory projections are to the ipsilateral hemisphere [10] and the absence of direct contralateral projections within the olfactory system is thought to be why lateralized responses to odours have been obtained in chicks [25],
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Table 1 Number of chicks head shaking and pecking in response to different odorants Odorant concentration (% v/v)
Head shaking
Pecking
Experiment 1 Eugenol 0 0.01 0.1 1.0 10.0 100.0
LN
RN
LN
RN
1 1 2 1 2 1
2 2 2 2 2 5
5 5 4 4 4 5
6 6 6 5 6 6
iso-Amyl acetate 0 0.01 0.1 1.0 10.0 100.0
2 1 2 2 5 6
2 1 1 2 4 6
5 6 6 5 5 6
5 6 6 6 6 5
Experiment 2 Unscented Eugenol (100.0) iso -amyl acetate (100.0)
LN 3 1 7
RN 2 7 7
LN 8 7 8
RN 8 8 6
Experiment 3 Eugenol 0 1.0 10.0 100.0
Oc
BN
Oc
BN
2 3 2 2
2 3 2 6
6 5 6 6
6 6 6 6
2 2 3 4
3 3 5 6
6 6 6 6
6 5 4 6
iso-Amyl acetate 0 1.0 10.0 100.0
Fig. 2. Mean (9S.E.M.) head shaking (a) and pecking responses (b) of chicks tested with unscented stimuli or stimuli scented with 100% (v/ v) concentrations of eugenol or iso -amyl acetate. Three separate groups of chicks (n 8 per group) were tested with either the left (right nostril in use, RN, j) or right nostril (left nostril in use, LN, I) occluded during testing. *P B 0.05, paired t -test.
LN, left nostril in use; RN, right nostril in use; BN, both nostrils in use; Oc, both nostrils occluded. Sample size: Experiments 1 and 3 (n 6 per odour); Experiment 2 (n 8 per odour).
rats [13] and humans [28]. By contrast, there have been few investigations into the central connections of the chicks’ trigeminal system, although the peripheral branches of this nerve have been described [6]. In the pigeon, however, the principal trigeminal nucleus, which receives sensory input from the various branches of the trigeminal nerve, projects monosynaptically and bilaterally to the telencephalon (nucleus basalis), bypassing the thalamus [27]. The main branch of the trigeminal nerve thought to be involved in the detection of odours in birds is the ophthalmic branch [18,26]. The anterior portion of the nasal cavity is supplied by the ethmoid nerve (a branch of the ophthalmic nerve), whereas the posterior portion of the nasal cavity is supplied by the nasopalatine nerve (a branch of the maxillary nerve), and these are also thought to carry chemosensory information [19]. The results of Experiment 3 rule out the involvement of receptors outside the nasal cavity, such as taste receptors or free nerve endings of the trigeminal nerve located in
Fig. 3. Mean (9S.E.M.) head shaking (a, b) and pecking (c, d) responses of chicks tested with various concentrations of (a, c) eugenol and (b, d) iso -amyl acetate. The chicks (n 6 per group) were either controls (both nostrils in use, BN, k) or had both nostrils occluded (Oc, m). They were tested repeatedly and presented with stimuli (bead colour and odorant concentration) in a Latin-square design. #P B 0.10, *P B 0.05, unpaired t -test.
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the mucous membranes of the eyes and oral cavity, as well as the outer surface of the beak. Thus, chicks appear to detect these volatile substances primarily by the olfactory system, but we can not rule out involvement of intranasal trigeminal chemoreception. Trigeminal chemoreception is thought to warn an animal against harmful or irritating chemical stimuli. Indeed, trigeminal responses to suprathreshold concentrations are often reflexive and protective [11]. However, the trigeminal system can be stimulated by non-irritating stimuli such as phenylethyl alcohol [20] and trigeminal stimulation can decrease the excitability of the olfactory bulb in the absence of irritating stimuli [22,23]. Thus, there are similarities between the two chemoreceptive systems and they are clearly inter-related. An alternate (and equally likely) explanation for the present findings may be that they are due to the way in which the chick interprets and learns about the odour, or even to lateralization depending on the brain regions which receive input from the trigeminal and olfactory systems, rather than whether either trigeminal or olfactory receptors or both are stimulated by odorants per se. For example, Vallortigara and Andrew [25] found that chicks with previous binarial exposure to a familiar stimulus scented with clove oil show a right nostril bias for that stimulus when they are tested, with one naris occluded, at 3 days of age in a laneway. By contrast, pigeons which have been habituated to amyl acetate using one nostril are still habituated to that odour when it is presented to the contralateral nostril [12]. However, these authors did not indicate when the left or right nostril was used, despite the fact that these birds had had the anterior commissure sectioned. Thus, in the former study [25], the memory for an odour associated with an object on which the chick has imprinted may be stored in a lateralized way, such that the right but not the left nostril has direct access to this memory, whereas in the latter study [12] the memory for an odorant which has a less complex association, i.e. not associated with food, parents or siblings, may be accessible equally by the left and right nostril. The perceived intensity of a range of odorants including, iso -amyl acetate and eugenol, has been tested in humans [9]. The responses of normal subjects (with the olfactory and trigeminal nerves intact) were compared with subjects that did not have an olfactory nerve but had a trigeminal nerve (referred to as anosmics). In this study eugenol was detected by 7% of anosmics (mean intensity /0.13) and by 100% of normal subjects (mean intensity /5.2), whereas iso -amyl acetate was detected by 100% of the anosmics (mean intensity / 6.67) and 100% of normal subjects (mean intensity / 6.67). A strong negative correlation was found between the perceived intensity of the odorant and its perceived pleasantness in normal as well as anosmic subjects.
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Therefore, it may be that chicks also respond to a higher intensity odorant, such as iso -amyl acetate, as being less pleasant than a lower intensity odorant, such as eugenol. However, behavioural screening of odours by chicks that have had their trigeminal or olfactory nerves sectioned has as yet not been tested. The task used in the present study relies on both visual and olfactory cues and it is likely that there is an interaction between colour and odour in the chick’s behavioural responses. However, we used a range of six differently coloured beads in the present study for both eugenol and iso -amyl acetate and in a balanced design. Hence, bead colour was not the determining factor of the results that we report here. In this study we have looked primarily at detection of volatile chemicals. Previous evidence for olfactory lateralization in chicks indicates that the right hemisphere plays a greater role than the left hemisphere when recognition, memory and preferences are involved. The present results are consistent with the hypothesis that there is lateralization to olfactory cues. We suggest that, when potentially irritating chemicals are involved, at least those which stimulate largely the trigeminal system, the attention of both hemispheres is engaged, possibly for a more rapid response to a potential source of harm.
Acknowledgements The authors thank Dr A.N.B. Johnston for valuable comments on the manuscript. THJB was supported by a University of New England Research Scholarship and the research formed part of his research towards a PhD degree.
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[7] Burne THJ, Rogers LJ. Responses to odorants by the domestic chick. Physiol Behav 1996;60:1441 /7. [8] Burne THJ, Rogers LJ. Relative importance of odour and taste in the one-trial passive avoidance learning bead task. Physiol Behav 1997;62:1299 /302. [9] Doty RL, Brugger WE, Jurs PC, Orndorff MA, Snyder PJ, Lowry LD. Intranasal trigeminal stimulation from odorous volatiles: psychometric responses from anosmic and normal humans. Physiol Behav 1978;20:175 /85. [10] Eslinger PJ, Damasio AR, Van Hoesen GW. Olfactory dysfunction in man: anatomical and behavioral aspects. Brain Cognit 1982;1:259 /85. [11] Farbman AI. Cell biology of olfaction. Cambridge: Cambridge University Press, 1992, 296 pp. [12] Gagliardo A, Teysse`dre A. Interhemispheric transfer of olfactory information in homing pigeon. Behav Brain Res 1988;27:173 /8. [13] Heine O, Galaburda AM. Olfactory asymmetry in the rat brain. Exp Neurol 1986;91:392 /8. [14] Jones RB, Roper TJ. Olfaction in the domestic fowl: a critical review. Physiol Behav 1997;62:1009 /18. [15] Rogers LJ. The development of brain and behaviour in the chicken. Wallingford: CAB International, 1995, 288 pp. [16] Rogers LJ. Behavioral, structural and neurochemical asymmetries in the avian brain: a model system for studying visual development and processing. Neurosci Biobehav Rev 1996;20:487 /503. [17] Rogers LJ, Andrew RJ, Burne THJ. Light exposure of the embryo and development of behavioural lateralization in chicks: I. Olfactory responses. Behav Brain Res 1998;97:195 /200.
[18] Sieck MH, Wenzel BM. Electrical activity of the olfactory bulb of the pigeon. Electroenceph Clinical Neurophysiol 1969;26:62 /9. [19] Silver WL. The common chemical sense. In: Finger TE, Silver WL, editors. Neurobiology of taste and smell. New York: Wiley, 1987:65 /87. [20] Silver WL, Arzt AH, Mason JR. A comparison of the discriminatory ability and sensitivity of the trigeminal and olfactory systems to chemical stimuli in the tiger salamander. J Comp Physiol A 1988;164:55 /66. [21] Slotnick BM, Schoonover FW. Olfactory pathways and the sense of smell. Neurosci Biobehav Rev 1992;16:453 /72. [22] Stone H, Carregal EJA, Williams B. The olfactory-trigeminal response to odorants. Life Sci 1966;5:2195 /201. [23] Stone H, Williams B, Carregal EJA. The role of the trigeminal nerve in olfaction. Exp Neurol 1968;21:11 /9. [24] Vallortigara G, Andrew RJ. Lateralization of response by chicks to change in a model partner. Anim Behav 1991;41:187 /94. [25] Vallortigara G, Andrew RJ. Olfactory lateralization in the chick. Neuropsychologia 1994;32:417 /23. [26] Walker JC, Walker DB, Tambiah CR, Gilmore KS. Olfactory and nonolfactory odor detection in pigeons: elucidation by a cardian acceleration paradigm. Physiol Behav 1986;38:575 /80. [27] Yasuda M. Nerve regulation of the food and water intake in chicken. In: Ookawa T, editor. The brain and behavior of the fowl. Tokyo: Japan Scientific Societies Press, 1983:185 /211. [28] Zatore RJ, Jones-Gotman M. Right nostril advantage for discrimination of odors. Percept Psychophys 1990;47:526 /31.
Research report
Chemosensory input and lateralization of brain function in the domestic chick Thomas H.J. Burne *, Lesley J. Rogers Centre of Neuroscience and Animal Behaviour, School of Biological, Biomedical and Molecular Sciences, The University of New England, Armidale 2351, Australia Received 15 October 2001; received in revised form 2 January 2002; accepted 2 January 2002
Abstract One-day old domestic chicks (Gallus gallus domesticus ) show concentration-dependent behavioural responses to olfactory cues. In the present study we investigated the lateralized olfactory responses of 1-day-old chicks to the odours of eugenol and iso -amyl acetate. In experiment 1 different concentrations of each odour were presented in repeated trials to chicks housed individually. The odours were presented together with a small coloured bead at which the chick pecked. When tested with the highest concentration of eugenol (100% v/v), the chicks demonstrated more head shaking when their left nostril was occluded (RN; right nostril in use) than when their right nostril was occluded (LN; left nostril in use). No such lateralization occurred in response to iso -amyl acetate. This result was confirmed in a second experiment in which the chicks were tested with unscented stimuli, 100% eugenol and 100% iso amyl acetate. In experiment 3 we found that occluding both the chicks’ nostrils abolished the head shaking response to eugenol and to iso amyl acetate. Thus, the chicks’ head shaking responses to the odorants eugenol and iso -amyl acetate are mediated primarily by inputs from within the nasal cavity, and not by oral or occular inputs. The present results are consistent with the hypothesis that there is lateralization to olfactory cues and that it is dependent on the involvement of receptors inside the nasal cavity. We suggest that differences in lateralized olfactory responses to different odours are affected by the relative involvement of intranasal olfactory and trigeminal chemoreceptors. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Domestic chicks; Chemosensory input; Lateralized olfactory responses; Olfaction
1. Introduction Differential use of the forebrain hemispheres by the chick has been established largely in response to visual cues [2 /4,16,24]. Studies in which chicks have been imprinted on an object or presented with a small bead and tested monocularly show specialized roles of the left and right hemispheres. It has been shown that the left hemisphere assigns stimuli to categories for considered responses, whereas the right hemisphere controls more immediate responses and attends to spatial cues [2,3,5,24].
* Corresponding author. Present address: Laboratory of Cognitive and Developmental Neuroscience, The Babraham Institute, Cambridge CB2 4AT, UK. Tel.: 44-1223-496-435; fax: 44-1223496-028 E-mail address: [email protected] (T.H.J. Burne).
The domestic chick uses odours in the formation of attachments (see [14] for review) and there is evidence that the hemispheres may be lateralized for controlling responses to a familiar odour. Vallortigara and Andrew [25] exposed chicks to clove oil odour contained in a small cylinder suspended in the home cage. On day 3 posthatching, each chick was tested in a runway with a cylinder containing the familiar odour suspended at one end and a similar cylinder not scented with clove oil at the other end. The chicks were tested with either the left or right nostril occluded with wax. Those with the left nostril occluded (right nostril in use; RN) chose to approach the clove oil scented stimulus in preference to the unscented stimulus. Chicks with the right nostril occluded (left nostril in use; LN) made no choice between the two stimuli. In another experiment Vallortigara and Andrew [25] reared chicks with an unscented cylinder and tested them in a laneway with a scented cylinder at one end (1
0166-4328/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 2 ) 0 0 0 0 9 - 8
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ml of n -amyl acetate, 1 ml of amyl acetate or 1 ml of orange oil) and an unscented cylinder at the other end to examine the chicks’ response to a novel odour. They found that RN chicks approached the familiar-unscented cylinder (so avoiding the novel-scented cylinder), whereas LN chicks approached both stimuli at random. Thus, RN (but not LN) chicks show a lateralized avoidance response to a novel odour, as well as a lateralized approach response to a familiar odour. Vallortigara and Andrew [25] also presented clove oil to chicks within a small peckable target, which was a grey, metal box containing a clean piece of cotton soaked with five drops of clove oil. The odour could diffuse out of the box through small holes in its walls. At test, 3-day-old male chicks, which had been reared with an unscented table tennis ball suspended in their cage, were presented with odour in four consecutive 10 s trials with either the left or right nostril occluded. A greater percentage of RN chicks (/70%) compared to LN chicks shook their heads on the first trial, indicating that they had detected the clove oil. Clove oil is believed to be a pure odorant that stimulates olfactory receptors in the nasal epithelium only and not the taste receptors or the free nerve endings of the trigeminal nerve in the oral cavity [9]. Occlusion of a nostril confines odour stimulation to the epithelium of the unoccluded nostril as the nasal cavities of chicks are separated by a cartilaginous septum [15]. The olfactory epithelium in each nasal cavity projects, via the olfactory nerve, to its ipsilateral olfactory bulb only [10], whereas the trigeminal inputs from each nasal cavity go to both hemispheres. Hence, unilateral stimulation of one nostril would lead to processing trigeminal inputs by both forebrain hemispheres due to stimulation of trigeminal receptors, but stimulation of olfactory receptors would involve primarily the ipsilateral forebrain hemisphere. Lateralized responses to odorants might be more likely to occur within the olfactory system, with its predominantly ipsilateral connections, rather than the trigeminal system. Thus, the finding that a preference for the familiar odour is expressed by chicks using the right but not the left nostril could be interpreted as indicating that the right hemisphere alone is specialized to process olfactory inputs and/or to control responses to odours. However, ipsilateral connections do not, in themselves, imply lateralization, unless the hemispheres which receive these projections process that information differentially. We have developed a behavioural task in which newly hatched chicks are allowed to peck at a small bead in the presence of an odorant [7]. The number of pecks directed at the bead and the amount of head shaking (a rapid, lateral movement of the head) that occurs during brief (10 s) trials is measured. Using this task we have shown that 1-day-old chicks demonstrate increased
amounts of head shaking and decreased amounts of pecking to increasing concentrations of some odorants, such as iso -amyl acetate and eugenol (eugenol is the primary component of clove oil and eugenol smells like clove oil to humans). We chose to use iso -amyl acetate in this experiment because it has been used widely in experiments in both mammals [14,21] and birds [26], and we have previously looked in detail at chicks responses to this odorant using this task [7]. All the above olfactory tasks [7,25] required the chick to process visual and olfactory input and thus the lateralization may reflect hemispheric specialization for the integration of visual and olfactory information. Rogers et al. [17] showed that head shaking to clove oil occurs for RN only when the bead is blue and not when it is red. Thus, there is an interaction between visual and olfactory cues. It appears that the visual cues override olfactory cues in the left hemisphere (LN condition) and that the left hemisphere ignores olfactory cues when the visual cues are very attractive, as in the case of the blue bead. The main question addressed in the experiments reported here is whether the lateralization of head shaking response occurs with eugenol and iso -amyl acetate, and over a range of doses. We predicted (based on previous findings [25]) that chicks using their left nostril (LN) would not shake their head, whereas RN chicks would, following the presentation of a range of concentrations of eugenol or iso -amyl acetate. To confirm the results of Experiment 1 we conducted a second experiment in which we presented chicks tested either using the LN or RN with either unscented stimuli or a single concentration of either eugenol or iso -amyl acetate. To rule out the involvement of receptors outside the nasal cavity in the response to eugenol and iso -amyl acetate we conducted a third experiment in which chicks were tested with both nostrils occluded.
2. Methods 2.1. Animals and housing Seventy-two chicks were used in this study, a separate group of 24 chicks being used in each of the three experiments. Fertile White Leghorn /Australorp eggs (Barter and Sons, Huntingwood, Australia) were incubated for the first 17 days in a forced-draft, turning incubator (Multiquip, Austral, Australia). The incubator was maintained at 37 /38 8C and 80% relative humidity. Viable eggs were transferred to a hatching incubator maintained at 37 /38 8C and 95% relative humidity from day 17 of incubation until 18 h posthatching. An illuminated heat lamp provided heat and light (100 /200 lx, depending on the position of the egg on the hatching tray). Eighteen hours after hatching
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each chick was housed individually in a clean grey metal cage (21 /21 /31 cm) on a layer of paper toweling and situated on a three-tier rack. The home cages consisted of galvanized steel on three sides and transparent plastic on the other. Warmth and illumination were provided by 40-W pearlescent bulbs and the temperature within the home cage was maintained at 29 /33 8C. Feeding and drinking were encouraged by sprinkling 5 g of chick starter mash (Fielders, Tamworth, Australia) in front of the chick and by dipping its beak into a water drinker located at the rear of the home cage. Thereafter the chicks remained undisturbed for 2 /6 h before they were tested. 2.2. Testing stimuli The test stimulus used has been described previously [7,8]. Briefly, it consisted of a coloured plastic bead (4 mm diameter) affixed to a white opaque 500 ml COBAS plastic sample cup which was attached to the end of a 250 mm long glass rod (4 mm outside diameter). The chicks were presented with the odour of eugenol or iso amyl acetate by applying 10 ml of the odorant to cotton wool contained in the sample cup. Different concentrations of odorant were prepared by making serial dilutions in ethanol and applying 10 ml of each dilution of solution to the cotton wool. We used the following dilutions; 0, 0.01, 0.1, 1, 10 or 100% (v/v). The walls of the sample cup were perforated with 15 evenly spaced 0.5 mm diameter holes to allow for adequate dispersal of odorant. We chose to present a range of bead colours, including yellow, light green, dark green, light blue, dark blue and red. This method was used to avoid habituation of pecking, which occurs rapidly in the chick [1], over repeated trials. A white bead was used during the training trials only. 2.3. Testing conditions Chicks were tested in a separate grey metal cage, referred to as the testing cage, that had the same dimensions as the home cage. Their behaviour was recorded on video tape by a centrally situated video camera located directly above the testing cage and connected to an external monitor. The front of the cage consisted of a metal sheet with 9 holes (7 mm diameter) spaced evenly in two rows approximately 50 and 70 mm from the floor of the cage. The testing room was visible through these holes and they served to direct the chick’s attention towards the front of the cage. The testing stimulus was introduced into the testing cage through a tenth, centrally placed hole (18 mm diameter). The testing cage was illuminated with a single 40-W pearlescent bulb. To prevent the chick from seeing the experimenter, the image from the monitor was used to guide the bead to a central position 5 mm from the tip of
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the chick’s beak. Although this testing procedure meant that the chicks received extra handling (they were carried gently ca. 1.5 m from the home cage to the testing cage), it enabled standardization of the test environment. 2.4. Occluding nostrils The chicks had one or other nostril occluded temporarily with an unscented wax preparation (Tender Beauty Products, Newcastle, Australia) 10 min before testing. The wax preparation was relatively easy to apply and, when applied to the chick’s beak, remained in place for at least 24 h. The wax was softened by placing it into a glass vial immersed in warm water. It was then applied to one or other of the chick’s nostrils using a small metal spatula such that it completely covered the external opening and a small portion of the surrounding beak. The application of a thin layer of the wax (less than 2 mm) to the nostrils did not obscure the chick’s vision. Some of the chick’s initially attempted to remove the wax plug by wiping their bill on the floor of the cage and scratching at their bill with their feet. However, none of the wax plugs became dislodged. In experiment 1 either the chick’s left (RN: right nostril in use) or right nostril (LN: left nostril in use) was occluded. In experiment 2 half of the chicks had their left nostril occluded in the first testing trial and their right nostril occluded in the second testing trial, and the remainder had the reverse condition (right nostril occluded in the first trial and left nostril occluded in the second trial). In experiment 3 half of the chicks had both nostrils occluded (Oc), and the remainder had unobstructed nostrils (BN: both nostrils in use). 2.5. Testing procedure Testing began when each chick was 20 /24 h posthatching and involved presentation of a white bead attached to an unscented sample cup for two 20-s trials in the testing cage. These ‘training trials’ were used to familiarize the chicks with the testing apparatus. In Experiment 1 four separate groups of six chicks were tested repeatedly over six trials with 0, 0.01, 0.1, 1, 10 or 100% (v/v) concentrations (presented in a Latin-square design) of one or other of the odorants (eugenol or iso amyl acetate). In Experiment 2 three separate groups of eight chicks were repeatedly tested over two trials with either unscented stimuli or 100% (v/v) concentrations of one of the odorants (eugenol or iso -amyl acetate). In Experiment 3 four separate groups of six chicks were repeatedly tested over four trials with 0, 1, 10 or 100% (v/v) concentrations of one of the odorants (eugenol or iso -amyl acetate). In Experiments 1 and 3 the colour of bead used in each test was allocated in a Latin-square design. The stimuli were presented to the chick in 10-s
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trials separated by 10-min intervals during which the chick was returned to its home cage. In Experiment 2 a chrome, coloured bead was used in both trials and the inter-trial interval was increased to 20 min so that the wax occluding one of the chicks’ nostrils could be removed and the contralateral nostril occluded. The handling procedure may have altered the chicks’ level of responding, although it was noted that the chicks were not unduly stressed and were, in the main, motivated to peck at the bead during each trial. Also, any effects of handling were consistent between treatments. In all of the trials the number of pecks at the bead and the number of bouts of head shaking were recorded during each of the 20 s (training) and 10 s (testing) periods using video tapes. The number of pecks directed at the glass tube or sample cup was not scored; more than 95% of pecks were directed at the bead. A bout of head shaking was scored when at least two rapid alternating lateral movements of the head occurred within 0.1 s. A separate bout of head shaking was scored after the head remained stationary for 0.4 s. Although lateralized responses for head shaking and not pecking have been reported previously [17,25] both data sets for pecking and head shaking are presented.
3. Results
main effect of the factor ‘Nostril’ (F1,20 /0.5, P /0.5) or ‘Colour’ (F5,100 /0.8, P /0.6) on head shaking and the interaction of the main effects of Odour, Nostril and Dose failed to reach significance (F5,100 /1.7, P /0.15). Inspection of the data presented in Fig. 1 suggests that any effects of Nostril on head shaking were restricted to the highest concentration of eugenol presented. Analysis of the head shaking responses to 100% concentrations revealed a significant main effect of Odour (F1,20 /7.4, P /0.01) and a signifcant interaction of Odour and Nostril (F1,20 /5.6, P / 0.03). There was a significant difference between the head shaking response of LN and RN chicks to the highest concentration of eugenol presented (100% v/v; t10 /3.1, P /0.01; Fig. 1a). The number of bouts of head shaking by RN chicks presented with 100% eugenol was also significantly higher than that given to the control stimulus (0%; t10 /3.1, P /0.01). This contrasts with the responses of LN chicks, which had similar head shaking scores for all concentrations of eugenol presented. There were no significant differences between the head shaking responses of LN and RN chicks to any concentration of iso -amyl acetate presented (Fig. 1b), even though concentration /response curves were evident for chicks tested with LN and RN. There was no significant effect of Colour on the head shaking data (F5,100 /0.8, P / 0.6). There was a non-significant tendency for an effect of the main factor Colour on the pecking scores (F5,100 / 2.2, P /0.06). Overall chicks pecked highest at light blue coloured beads (6.39/0.7) and least at yellow coloured beads (3.49/0.7), with an intermediate number of pecks given to the remaining differently coloured beads. There were no significant effects of the other main factors or their interactions on the pecking scores (P / 0.10). The proportion of chicks responding at each dose of each odorant is shown in Table 1. The number of chicks pecking did not vary in response to the presentation of each concentration of odorant. By contrast the number of LN and RN chicks head shaking was highest in response to the two highest concentrations of iso -amyl acetate presented (10 and 100%). The number of RN chicks head shaking was highest at the highest concentration of eugenol presented (100%), whereas the number of LN chicks head shaking did not increase in response to the highest concentration of eugenol presented (100%).
3.1. Experiment 1
3.2. Experiment 2
There was a significant main effect of the factor ‘Dose’ on the chicks’ head shaking responses (F5,100 / 14.1, P B/ 0.001). There was also a significant effect of the interaction between the main factors ‘Odour’ and Dose (F5,100 /5.2, P B/ 0.001). There was no significant
There was a significant effect of Odour on the chicks head shaking (F2,21 /9.7, P B/0.01) and pecking responses (F2,21 /4.8, P /0.02). As can be seen in Fig. 2 the chicks had high pecking and low head shaking scores to unscented stimuli and low pecking and high head
2.6. Statistical analysis The data were square-root transformed (n/1) and analysed using repeated measures ANOVA or one-way ANOVA. In Experiment 1 the main factors analysed were Odour (eugenol or iso -amyl acetate), Nostril (LN or RN) and the repeated measures were fitted on the factors Dose (0. 0.01, 0.1, 1, 10 or 100% concentrations) and Colour (red, yellow, light blue, dark blue, light green and dark green). In Experiment 2 the main factor analysed was Odour (unscented, eugenol or iso -amyl acetate) and the repeated measure was fitted on the factor Nostril (LN or RN). In Experiment 3 the main factors analysed were Odour (Eugenol or iso -amyl acetate) and Nostril (occluded or unobstructed), and the repeated measure was fitted on the factor Dose (0, 1, 10 and 100). Post-hoc analysis was performed with either paired or unpaired t -tests. Statistical values with a probability value (P ) of B/0.05 were considered to indicate significant differences between groups.
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Fig. 1. Mean (9S.E.M.) head shaking (a, b) and pecking (c, d) responses of chicks tested with various concentrations of (a, c) eugenol or (b, d) iso amyl acetate. Four separate groups of chicks (n 6 per group) had either the left (right nostril in use, RN, m) or right nostril (left nostril in use, LN, k) occluded during testing. They were tested repeatedly and presented with stimuli (bead colour and odorant concentration) in a Latin-square design. *P B 0.05, paired t -test.
shaking scores to iso -amyl acetate. The chicks had intermediary head shaking and pecking responses to eugenol. There was a significant interaction of the factors Nostril and Odour (F2,21 /8.2, P /0.002) on the head shaking scores but not on the pecking scores (F2,21 /0.4, P /0.7). Post-hoc analysis with paired ttests indicated that chicks had higher head shaking scores when they were using their RN, rather than their LN, in response to eugenol (t7 /5.8, P B/0.01) but not to iso -amyl acetate (t7 /0.3, P /0.8) or unscented stimuli (t7 /0.6, P /0.6). This confirms the results found in Experiment 1. 3.3. Experiment 3 There was a significant main effect of Nostril (F1,20 / 10.4, P /0.004) and Odour (F1,20 /5.8, P /0.03) on the head shaking response. The interaction between Nostril (BN or Oc) and Odour failed to reach significance (F1,20 /3.5, P /0.08). There was a significant main effect of Dose on the head shaking scores (F3,60 /7.0, P B/0.001). It can be seen in Fig. 3 that there appeared to be a trend for the head shaking scores of Oc chicks to increase with increasing concentrations of iso -amyl acetate and eugenol. Chicks tested with both nostrils in use (BN) had significantly higher head shaking scores than chicks with both nostrils occluded (Oc) at the 100% concentration for iso -amyl acetate (t10 /4.0, P B/0.01). There was a tendency for BN chicks to shake their head more than Oc chicks to 10% iso -amyl acetate (t10 /1.9, P /0.09) and 100% eugenol (t10 /2.1, P /0.06).
There was a significant main effect of Nostril on the pecking response (F1,20 /8.5, P /0.009); Oc chicks had higher pecking scores, overall, than BN chicks. BN chicks pecked significantly less than Oc chicks to 10% (t10 /3.5, P /0.005) and 100% iso -amyl acetate (t10 / 5.8, P B/0.001). There were no significant effects of the other main factors (Dose or Odour) or their interactions on the pecking scores.
4. Discussion The main result of this study was that there was a lateralization in head shaking in response to the highest concentration of eugenol but not of iso -amyl acetate. This result was confirmed in a second experiment in which only the highest concentration of eugenol and iso amyl acetate were presented. The results from a third experiment, in which the chicks were tested with both nostrils occluded, ruled out the involvement of extra nasal receptors, such as those from oral or occular inputs in association with the odours tested. Therefore, 1-day-old chicks do not show a right nostril advantage for the perception of all odorants. One possible explanation for the present findings is the relative involvement of the olfactory and trigeminal receptors within the nasal cavity. The principle olfactory projections are to the ipsilateral hemisphere [10] and the absence of direct contralateral projections within the olfactory system is thought to be why lateralized responses to odours have been obtained in chicks [25],
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Table 1 Number of chicks head shaking and pecking in response to different odorants Odorant concentration (% v/v)
Head shaking
Pecking
Experiment 1 Eugenol 0 0.01 0.1 1.0 10.0 100.0
LN
RN
LN
RN
1 1 2 1 2 1
2 2 2 2 2 5
5 5 4 4 4 5
6 6 6 5 6 6
iso-Amyl acetate 0 0.01 0.1 1.0 10.0 100.0
2 1 2 2 5 6
2 1 1 2 4 6
5 6 6 5 5 6
5 6 6 6 6 5
Experiment 2 Unscented Eugenol (100.0) iso -amyl acetate (100.0)
LN 3 1 7
RN 2 7 7
LN 8 7 8
RN 8 8 6
Experiment 3 Eugenol 0 1.0 10.0 100.0
Oc
BN
Oc
BN
2 3 2 2
2 3 2 6
6 5 6 6
6 6 6 6
2 2 3 4
3 3 5 6
6 6 6 6
6 5 4 6
iso-Amyl acetate 0 1.0 10.0 100.0
Fig. 2. Mean (9S.E.M.) head shaking (a) and pecking responses (b) of chicks tested with unscented stimuli or stimuli scented with 100% (v/ v) concentrations of eugenol or iso -amyl acetate. Three separate groups of chicks (n 8 per group) were tested with either the left (right nostril in use, RN, j) or right nostril (left nostril in use, LN, I) occluded during testing. *P B 0.05, paired t -test.
LN, left nostril in use; RN, right nostril in use; BN, both nostrils in use; Oc, both nostrils occluded. Sample size: Experiments 1 and 3 (n 6 per odour); Experiment 2 (n 8 per odour).
rats [13] and humans [28]. By contrast, there have been few investigations into the central connections of the chicks’ trigeminal system, although the peripheral branches of this nerve have been described [6]. In the pigeon, however, the principal trigeminal nucleus, which receives sensory input from the various branches of the trigeminal nerve, projects monosynaptically and bilaterally to the telencephalon (nucleus basalis), bypassing the thalamus [27]. The main branch of the trigeminal nerve thought to be involved in the detection of odours in birds is the ophthalmic branch [18,26]. The anterior portion of the nasal cavity is supplied by the ethmoid nerve (a branch of the ophthalmic nerve), whereas the posterior portion of the nasal cavity is supplied by the nasopalatine nerve (a branch of the maxillary nerve), and these are also thought to carry chemosensory information [19]. The results of Experiment 3 rule out the involvement of receptors outside the nasal cavity, such as taste receptors or free nerve endings of the trigeminal nerve located in
Fig. 3. Mean (9S.E.M.) head shaking (a, b) and pecking (c, d) responses of chicks tested with various concentrations of (a, c) eugenol and (b, d) iso -amyl acetate. The chicks (n 6 per group) were either controls (both nostrils in use, BN, k) or had both nostrils occluded (Oc, m). They were tested repeatedly and presented with stimuli (bead colour and odorant concentration) in a Latin-square design. #P B 0.10, *P B 0.05, unpaired t -test.
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the mucous membranes of the eyes and oral cavity, as well as the outer surface of the beak. Thus, chicks appear to detect these volatile substances primarily by the olfactory system, but we can not rule out involvement of intranasal trigeminal chemoreception. Trigeminal chemoreception is thought to warn an animal against harmful or irritating chemical stimuli. Indeed, trigeminal responses to suprathreshold concentrations are often reflexive and protective [11]. However, the trigeminal system can be stimulated by non-irritating stimuli such as phenylethyl alcohol [20] and trigeminal stimulation can decrease the excitability of the olfactory bulb in the absence of irritating stimuli [22,23]. Thus, there are similarities between the two chemoreceptive systems and they are clearly inter-related. An alternate (and equally likely) explanation for the present findings may be that they are due to the way in which the chick interprets and learns about the odour, or even to lateralization depending on the brain regions which receive input from the trigeminal and olfactory systems, rather than whether either trigeminal or olfactory receptors or both are stimulated by odorants per se. For example, Vallortigara and Andrew [25] found that chicks with previous binarial exposure to a familiar stimulus scented with clove oil show a right nostril bias for that stimulus when they are tested, with one naris occluded, at 3 days of age in a laneway. By contrast, pigeons which have been habituated to amyl acetate using one nostril are still habituated to that odour when it is presented to the contralateral nostril [12]. However, these authors did not indicate when the left or right nostril was used, despite the fact that these birds had had the anterior commissure sectioned. Thus, in the former study [25], the memory for an odour associated with an object on which the chick has imprinted may be stored in a lateralized way, such that the right but not the left nostril has direct access to this memory, whereas in the latter study [12] the memory for an odorant which has a less complex association, i.e. not associated with food, parents or siblings, may be accessible equally by the left and right nostril. The perceived intensity of a range of odorants including, iso -amyl acetate and eugenol, has been tested in humans [9]. The responses of normal subjects (with the olfactory and trigeminal nerves intact) were compared with subjects that did not have an olfactory nerve but had a trigeminal nerve (referred to as anosmics). In this study eugenol was detected by 7% of anosmics (mean intensity /0.13) and by 100% of normal subjects (mean intensity /5.2), whereas iso -amyl acetate was detected by 100% of the anosmics (mean intensity / 6.67) and 100% of normal subjects (mean intensity / 6.67). A strong negative correlation was found between the perceived intensity of the odorant and its perceived pleasantness in normal as well as anosmic subjects.
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Therefore, it may be that chicks also respond to a higher intensity odorant, such as iso -amyl acetate, as being less pleasant than a lower intensity odorant, such as eugenol. However, behavioural screening of odours by chicks that have had their trigeminal or olfactory nerves sectioned has as yet not been tested. The task used in the present study relies on both visual and olfactory cues and it is likely that there is an interaction between colour and odour in the chick’s behavioural responses. However, we used a range of six differently coloured beads in the present study for both eugenol and iso -amyl acetate and in a balanced design. Hence, bead colour was not the determining factor of the results that we report here. In this study we have looked primarily at detection of volatile chemicals. Previous evidence for olfactory lateralization in chicks indicates that the right hemisphere plays a greater role than the left hemisphere when recognition, memory and preferences are involved. The present results are consistent with the hypothesis that there is lateralization to olfactory cues. We suggest that, when potentially irritating chemicals are involved, at least those which stimulate largely the trigeminal system, the attention of both hemispheres is engaged, possibly for a more rapid response to a potential source of harm.
Acknowledgements The authors thank Dr A.N.B. Johnston for valuable comments on the manuscript. THJB was supported by a University of New England Research Scholarship and the research formed part of his research towards a PhD degree.
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