Neuroendocrine control of maternal behaviour in birds
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Maternal behaviour in humans and animals promotes the survival and future success of offspring. It is mainly controlled by the brain behaviour network in the hypothalamus. The behavioural and physiological changes which occur when an animal ‘becomes maternal’ are significant and include the cessation of reproductive behaviours and often a modulation of the stress response, energy balance and aggression. While the mammalian maternal brain has been extensively studied, much less is known in birds. This PhD project aimed to investigate the peptides and neuroendocrine pathways which govern the onset and maintenance of avian maternal care. In the mammalian brain, maternal behaviour and other social behaviours are regulated by the nonapeptides oxytocin and vasopressin, which are also involved in the stress response and water balance. The oxytocin and vasopressin orthologues in birds and reptiles, called mesotocin and vasotocin, are very similar in structure and perform similar roles. However, very few studies have focussed on their involvement in maternal care. In the experiments presented in this thesis, it was found that mesotocin mRNA expression was higher in the paraventricular nucleus (PVN) of hens rearing chicks than in laying hens as early as the first day of rearing, suggesting the involvement of mesotocin in rearing in the chicken. In a subdivision of the lateral bed nucleus of the stria terminalis (BnSTl), mesotocin mRNA expression was lower during incubation, compared to laying and rearing, while vasotocin mRNA expression was lower in both incubating hens and hens on the first day of rearing, compared to layers. This differed from the expected results, as data from previous studies suggested that mesotocin in the medial BnST (BnSTm) of birds promoted maternal behaviour. Even though, to the knowledge of the author, the lateral sector of this nucleus had not been separately examined in this context prior to this project, results were expected to be similar, as the medial and lateral BnST are closely related and often examined together. It was speculated that the changes in the BnSTl which were observed could be connected to a possible decrease in social interactions in chickens during incubation. Incubating hens very rarely leave the nest and actively discourage others from approaching it. This could theoretically lead to fewer interactions with their conspecifics. However, no formal measurement of sociality and social interactions was performed during this project and, since the chickens in the relevant experiment were housed in pairs with both birds often entering incubation at roughly the same time, it would have been difficult to judge what their behaviour would have been if the rest of the flock had been present. A measurement of social interaction should be included in further experiments investigating the significance of nonapeptide changes in the BnSTl during incubation. With regards to vasotocin, it was speculated that the lower mRNA expression during incubation in the BnSTl could be related to an attenuated stress response in maternal birds. This suggestion was based on previous studies in other avian and mammalian species which have shown that parental animals can have lower responsiveness to stress. However, the stress response was not measured in this study and future work is necessary to determine whether it is indeed attenuated in the chicken under these conditions. In order for maternal behaviour to take place, sexual behaviours need to be inhibited. Gonadal steroids, which control sexual behaviours, have been shown to interact with nonapeptides in the brains of mammals and birds, but these interactions are complex, context-dependent and not fully understood. The results of this project showed that acute treatment with certain sex steroids after a period of priming was capable of significantly increasing the expression of mesotocin or vasotocin mRNA in specific areas of the brain behaviour network. In both the PVN and BnSTl, testosterone but not its metabolite estradiol increased mesotocin expression, suggesting that the action of testosterone on mesotocin was direct. Vasotocin expression was increased only in the BnSTl by both testosterone and estradiol, suggesting that the action of testosterone on vasotocin in this brain area was likely achieved through estradiol, following the aromatisation of testosterone. Gonadotropin inhibitory hormone (GnIH) is produced in the hypothalamus and has a strong inhibitory effect on the reproductive axis. GnIH shows significant changes throughout the reproductive cycle in the brains of both mammals and birds. After examining the changes in GnIH throughout the hen reproductive cycle, it was found that hens on the fourteenth day of incubation had a significantly higher number of GnIH-immunoreactive cells in the PVN compared to laying hens. These results were in agreement with previous findings and suggested that GnIH may be involved in downregulating reproductive behaviours during incubation. The hormone prolactin is involved in maternal care in mammals and birds and it is important in incubation and rearing. It is known to be controlled by the dopaminergic system through the D1 dopamine receptor (D1, D1R), which promotes, and the D2 dopamine receptor (D2, D2R), which inhibits its expression. In this project, quantitative polymerase chain reaction (qPCR) was used to measure prolactin and D2 mRNA in the pituitary glands of hens throughout the reproductive cycle, from laying eggs through to the first day of rearing chicks. No changes in mRNA expression for either prolactin or D2 were observed, suggesting that changes in their mRNA expression in the pituitary gland are not crucial for the display of incubation and rearing. It is possible that other mechanisms contribute strongly to the increase in plasma prolactin during incubation. Apart from controlling prolactin, dopamine is also involved in maternal and other social behaviours, such as sexual behaviour, social approach, social attachment, social dominance and aggression. Other monoamines, including serotonin (5-hydroxytryptamine, 5-HT), noradrenaline and adrenaline have also been implicated in social interactions, including aggression and maternal behaviour. All of these monoamines are present in the raphe nucleus but, to the author’s knowledge, the changes in their concentrations in this brain area throughout the reproductive cycle from egg-laying through to chick-rearing had not been characterised in birds prior to this project. Liquid chromatography - mass spectrometry (LC-MS) was used to examine the concentrations of monoamines in the raphe nucleus of the female chicken throughout the reproductive cycle and test the hypothesis that they might be involved in maternal care in this species. No differences were found between groups for any of the examined monoamines, which included adrenaline, noradrenaline, dopamine, 5-HT, the dopamine precursor dihydroxyphenylacetic acid (DOPAC), the 5-HT precursor tryptophan and tryptophan’s metabolite hydroxyanthranilic acid. These results do not provide any evidence that monoamines in the raphe nucleus play a role in incubation or rearing in the chicken. However, as only monoamine content rather than release was measured, a role mediated by differences in release cannot be excluded. In addition, limitations of the experimental procedure mean that results from this experiment should be interpreted with caution. Many animals display negative responses to the young of their species when not in a maternal state but habituation to young individuals can often alter these behaviours and produce a maternal response. The display of maternal care induces c-fos (an immediate early gene, marker of neuronal activation) expression in brain areas controlling the behaviour. The effects of social stimulation with chicks vs adults were tested in Japanese quail. The change of species from chicken was necessary due to unforeseen issues at the Roslin Institute Poultry Unit which led to the loss of the existing colony of maternal chickens. Unfortunately, despite many efforts, the colony could not be replaced in time for the aforementioned experiment to be conducted on the original species of choice. Japanese quail were the best replacement species the author had access to. While they rarely incubate eggs in captivity, they do display rearing behaviour, in addition to also being precocial, like the chicken, and having physiology and brain organisation similar to chickens. It was found that adult female Japanese quail habituated to chicks for 6 days spent significantly more time in close proximity to novel chicks compared to novel adult individuals and stimulation with chicks caused greater c-fos expression than stimulation with a novel adult in a brain area related to maternal and other social behaviours (the PVN) and, surprisingly, an area related to sexual behaviour (the nucleus of the commisurae pallii, nCPa). What type of cells were activated remains unclear and sexual behaviour was not tested for. In contrast, c-fos expression was lower in the group presented with chicks in the raphe nucleus – an area known to be involved in maternal behaviour, as well as stress. It can be speculated that, after habituation, chicks may have induced the beginning of maternal behaviour in females (and therefore higher activation in PVN neurones). They may have also presented a less stressful stimulus than a new adult (inducing less activation in the raphe nucleus). However, full maternal behaviour was not observed within the scope of this experiment and stress was not measured. Therefore, further studies are necessary to examine the significance of these brain areas in interactions with chicks, including determining what type of neurones were being activated. This thesis presents an overview of the changes throughout the reproductive cycle in some major hormones of the brain behaviour network and examines their possible roles in maternal behaviour through specific brain nuclei. It provides evidence for the involvement of the mesotocin/vasotocin and GnIH systems in the control of maternal care in birds and sheds more light on the complex relationship between sex steroids and the brain nonapeptides. The findings presented here contribute to the areas of neuroscience, physiology, developmental biology and endocrinology.