Kevin D. Broad

Neural Control of Maternal Behaviour and Olfactory Recognition of Offspring

In terms of reproductive success the quality and duration of maternal care exhibited by any particular species is of paramount importance, and yet compared with the amount of research studying the control of reproductive cycles, sexual behaviour, and fertility, it has historically received considerably less attention. However, we are now beginning to understand how the brain is organised to mediate this complex behaviour and how its expression is orchestrated by different hormonal and neurochemical factors. This review summarises a series of neuroanatomical, electrophysiological, in vivo sampling and behavioural neuropharmacological experiments carried out in sheep. These have attempted to define the neural circuitry and hormonal neurotransmitter systems involved both in the control of maternal behaviour per se and in the selective olfactory recognition of lambs, which is the basis of an exclusive emotional bond between mother and offspring.

Facial and vocal discrimination in sheep

The ability of sheep, Ovis aries, to discriminate between sheep, humans and other animals on the basis of facial and vocal cues was tested in an enclosed Y-maze. Pairs of faces or voices were presented which had a clear differential significance for the sheep (i.e. sheep versus human, dog or an unfamiliar breed or species). Some Clun Forest and Dalesbred sheep could actively distinguish (>75% choice) between different faces, with the best performances being seen for sheep versus human faces (the sheep face being preferred to that of the human). Other animals showed pronounced position preferences in the maze whatever faces were shown although they approached sheep faster than other faces, suggesting that they could discriminate between them. Dalesbred, but not Clun Forest, sheep could also discriminate between sheep and human vocalizations alone in the maze. For both breeds, combining appropriate sight and sound stimuli did not significantly enhance performance although mismatching them reduced it. In a further experiment on animals that performed at more than 75% choice criterion in the maze, inverting the faces, turning them to profile or masking the eyes all significantly reduced performance in discriminating between sheep and human faces. In another experiment, Clun Forest ewes could also distinguish between the faces of male and female breed members. During anoestrus they preferred the faces of females and when they were in oestrus they preferred those of rams. Overall, these results suggest that sheep can use facial cues to discriminate between different species, breeds and male and female members of the same breed. They also show that discriminatory performance is influenced by orientation, the presence of eyes and, to some extent, vocal cues.

Changes in oxytocin immunoreactivity and mRNA expression in the sheep brain during pregnancy, parturition and lactation and in response to oestrogen and progesterone.

The effects of pregnancy, parturition and lactation and exogenous treatments with oestradiol and progesterone on oxytocin (OXY) immunoreactivity and gene expression in the sheep brain were investigated. Immunocytochemistry was used to demonstrate that increased OXY‐immunoreactivity occurred in cells of the paraventricular (PVN) and supraoptic nuclei (SON), the bed nucleus of the stria terminalis (BNST), the anterior commissural nuclei (ACN) and the periventricular part of the medial preoptic area (PvMP). Oxytocin immunoreactive terminals were also seen in the accessory olfactory nucleus, the glomerular and peri‐glomerular layers of the olfactory bulb, the lateral septum, the zona incerta and the pars compacta of the substantia nigra. Compared to ovariectomized and late pregnant animals, the intensity of immunoreactivity was increased in all of these oxytocinergic elements at parturition, during lactation and following exogenous treatment with oestradiol. The OXY‐immunoreactivity was also more intense in late pregnant animals compared to ovariectomized ones. Quantitative in situ hybridization histochemistry showed that cells in the PVN, SON, BNST and PvMP all showed significantly increased expression of OXY mRNA in animals at parturition and during lactation compared to late pregnant or ovariectomized animals. Expression levels in late pregnant animals were also significantly higher than in ovariectomized ones. Progesterone treatment significantly increased OXY mRNA in the PVN, SON, BNST and PvMP whereas oestradiol treatment was only effective in the PVN, BNST and PvMP. Combined treatment with these steroids did not significantly increase OXY mRNA levels in comparison with their administration alone.

Changes in Pro‐Opiomelanocortin and Pre‐proenkephalin mRNA Levels in the Ovine Brain during Pregnancy, Parturition and Lactation and in Response to Oestrogen and Progesterone

In the female sheep opioids act centrally to influence both oxytocin release and maternal behaviour. We have used in situ hybridization histochemistry to investigate the changes in mRNA expression of the two opioid precursor genes, proopiomelanocortin (POMC) and pre‐proenkephalin (PPE), in discrete hypothalamic nuclei as a function of pregnancy, parturition and lactation and following treatment with oestrogen and progesterone. Quantitative in situ hybridization histochemistry demonstrated that POMC mRNA expression in the arcuate nucleus (ARC) decreased at parturition and increased during lactation compared to late pregnant and ovariectomized animals. Oestradiol and progesterone treatments increased POMC mRNA expression compared to ovariectomized controls. Pre‐proenkephalin mRNA expression was quantified in three discrete hypothalamic nuclei, the ventromedial nucleus (VMN), the paraventricular nucleus (PVN) and the suprachiasmatic nucleus (SCN). In the VMN, PPE mRNA expression increased during lactation compared to late pregnancy and parturition. Expression levels during late pregnancy and parturition were decreased compared to ovariectomized animals. Oestradiol increased, and progesterone decreased, PPE mRNA levels compared to ovariectomized controls. Combined progesterone followed by oestrogen treatment produced significant increases in PPE mRNA expression. In the PVN, PPE expression increased at parturition compared to late pregnant, lactating and ovariectomized animals. Expression levels in late pregnant animals were decreased compared to lactating or ovariectomized ones. However, sex steroid treatment produced no changes in PPE expression in the PVN. No changes were observed in PPE mRNA expression in the SCN in response to any of the experimental conditions. This data shows that both POMC and PPE mRNA levels are altered in the sheep brain during pregnancy, parturition and lactation and in response to sex steroids, although the direction of the changes is not always the same and in the case of PPE only the VMN and PVN are affected. Levels of gene expression found following exogenous steroid treatment do not precisely mirror those found during pregnancy, parturition and lactation and this suggests that factors other than changing sex steroids levels are involved. In the context of maternal behaviour it is interesting to note that PPE mRNA expression increases at parturition in the PVN when oxytocin mRNA expression levels are also increased.

Placental protection of the fetal brain during short-term food deprivation

The fetal genome regulates maternal physiology and behavior via its placenta, which produces hormones that act on the maternal hypothalamus. At the same time, the fetus itself develops a hypothalamus. In this study we show that many of the genes that regulate placental development also regulate the developing hypothalamus, and in mouse the coexpression of these genes is particularly high on embryonic days 12 and 13 (days E12–13). Such synchronized expression is regulated, in part, by the maternally imprinted gene, paternally expressed gene 3 (Peg3), which also is developmentally coexpressed in the hypothalamus and placenta at days E12–13. We further show that challenging this genomic linkage of hypothalamus and placenta with 24-h food deprivation results in disruption to coexpressed genes, primarily by affecting placental gene expression. Food deprivation also produces a significant decrease in Peg3 gene expression in the placenta, with consequences similar to many of the placental gene changes induced by Peg3 mutation. Such genomic dysregulation does not occur in the hypothalamus, where Peg3 expression increases with food deprivation. Thus, changes in gene expression brought about by food deprivation are consistent with the fetal genomes maintaining hypothalamic development at a cost to its placenta. This biased change to gene dysregulation in the placenta is linked to autophagy and ribosomal turnover, which sustain, in the short term, nutrient supply for the developing hypothalamus. Thus, the fetus controls its own destiny in times of acute starvation by short-term sacrifice of the placenta to preserve brain development.

Increased BDNF and trk-B mRNA expression in cortical and limbic regions following formation of a social recognition memory.

Brain‐derived neurotrophic factor (BDNF) and its receptor, tyrosine receptor kinase (trk‐B), play important roles in neural plasticity, long‐term potentiation and memory formation. Sheep form a selective recognition memory for their lambs within 2 h of birth. Initially, this memory is exclusively based on olfactory cues; however, as it consolidates over a 12‐h recognition period it extends to incorporate visual cues. We investigated whether changes in BDNF and trk‐B mRNA expression occurred in both olfactory and visual processing systems at 4.5 h postpartum, 2–3 h after the behavioural manifestations of an olfactory recognition memory were found. Animals that formed a recognition memory showed increased BDNF mRNA expression in the inferior part of the temporal cortex, subfield CA1 of the hippocampus, the diagonal band, basolateral amygdala and the anterior cingulate, medial frontal, entorhinal and pyriform cortices. No increases were observed in either the olfactory bulbs or the dentate gyrus. Expression of trk‐B mRNA was significantly increased only in the medial temporal, entorhinal and pyriform cortices. These findings demonstrate that by 2–3 h following the initial formation of olfactory recognition memory there are BDNF/trk‐B‐mediated plasticity changes in brain areas involved in the consolidation of olfactory memory (the pyriform and entorhinal cortices). However, similar changes also occur in areas of the brain involved in visual memory, face and object recognition (the temporal cortex, entorhinal cortex, hippocampal subfield CA1 and basolateral amygdala), and in areas of the brain with integrative and attentional functions (the medial frontal and anterior cingulate cortices and diagonal band). This suggests that reorganization of neural circuits underlying the visual recognition of lambs or the integration of olfactory/visual information is occurring even at this time even though accurate behavioural recognition at this stage can only be made using olfactory cues.

The Role of Oxytocin Release in the Mediobasal Hypothalamus of the Sheep in Relation to Female Sexual Receptivity

In vivo microdialysis and retrodialysis were used to investigate the role of oxytocin (OXY) release in the mediobasal hypothalamus (MBH) of the ewe in the control of sexual receptivity. Initial experiments showed that OXY release was significantly increased in ovariectomized animals treated with progesterone and oestradiol when they were sexually receptive towards males and received intromissions. No such increases were seen during tests where the ewes were receptive but the males were prevented from achieving intromission. By contrast, OXY release was significantly reduced in tests where the ewes were not receptive to the male. In a second experiment artificial vaginocervical stimulation (VCS) was found to significantly increase OXY release when the animals were treated with oestradiol and this effect was potentiated by progesterone priming. OXY release in the MBH was not significantly altered by VCS in the presence of progesterone priming alone. Plasma OXY concentrations were significantly increased by VCS following all three hormone treatments but no one treatment was significantly more effective than another. Noradrenaline release in the MBH was only significantly increased following VCS when progesterone priming was given before oestradiol treatment. No effects of VCS on release of GABA, glutamate or dopamine were seen but their basal concentrations were significantly increased by the combined steroid treatment compared to oestradiol alone. In a third experiment it was found that OXY (10 μM) infused bilaterally into the MBH of receptive ewes, by retrodialysis, significantly decreased sexual receptivity and increased the release of noradrenaline and GABA. Finally, in a fourth experiment is was shown that multiple intromissions significantly reduced sexual receptivity.

Is right hemisphere specialization for face discrimination specific to humans

Patterns of neural activation during face recognition were investigated in sheep by quantifying altered c‐fos mRNA expression in situations where faces (sheep vs. human) can (faces upright) and cannot (faces inverted) be discriminated. Exposure to upright faces selectively increased expression significantly more in the right inferior temporal cortex than in the left, and active choice between upright faces additionally increased expression bilaterally in basal amygdala and hippocampus (CA1–4). Exposure to inverted faces did not lead to enhanced activation in the right inferior temporal cortex, amygdala or hippocampus but instead increased expression levels in the diagonal band of Broca, parietal and cingulate cortices. These results show that discrimination of upright faces in sheep preferentially engages the right temporal cortex, as it does in humans, and that performance of active choices between such faces may additionally involve the basal amygdala and hippocampus.

Increased Apoptosis During Neonatal Brain Development Underlies the Adult Behavioral Deficits Seen In Mice Lacking a Functional Paternally Expressed Gene 3 (Peg3)

Inactivation of the maternally imprinted, paternally expressed gene 3 (Peg3) induces deficits in olfactory function, sexual and maternal behaviors, oxytocin neuron number, metabolic homeostasis and growth. Peg3 is expressed in a number of developing hypothalamic and basal forebrain structures and is a component of the P53 apoptosis pathway. Peg3 inactivation in neuronal cell culture lines inhibits P53 mediated apoptosis, which is important in the early postnatal development and sexual differentiation of the brain. In this study, we investigated the effect of inactivating the Peg3 gene on the incidence of caspase 3 positive cells (a marker of apoptosis) in 4‐ and 6‐day postpartum mouse brain. Inactivating the Peg3 gene resulted in an increase in the incidence of total forebrain caspase 3 positive cells at 4 and 6 days postpartum. Increases in specific neuroanatomical regions including the bed nucleus of the stria terminalis, nucleus accumbens, caudate putamen, medial pre‐optic area, arcuate nucleus, medial amygdala, anterior cortical and posteriodorsal amygdaloid nuclei, were also observed. In wild‐type mice, sex differences in the incidence of caspase 3 positive cells in the medial amygdala, bed nucleus of the stria terminalis, nucleus accumbens, arcuate nucleus and the M2 motor cortex, were also observed. This neural sex difference was ameliorated in the Peg‐3 mutant. These findings suggest that the neuronal and behavioral deficits seen in mice lacking a functional Peg3 gene are mediated by increases in the incidence of early neonatal apoptosis in neuroanatomical regions important for reproductive behavior, olfactory and pheromonal processing, thermoregulation and reward.

Involvement of the medial prefrontal cortex in mediating behavioural responses to odour cues rather than olfactory recognition memory.

Sheep form an olfactory recognition memory for their lambs within 2 h of parturition and will subsequently reject the approaches of any strange lamb and protest vocally. In this study we report that following olfactory memory formation, ewes exposed to either their own or a strange lamb show c-fos mRNA expression in the medial frontal cortex, although levels of expression in the pyramidal output cell layer V were significantly higher in ewes that rejected strange lambs. Reversibly inactivating this region by the retrodialysis of the anaesthetic tetracaine before birth reduced aggressive motor responses towards lambs but not protest vocalisations. Similar treatment during the critical period for olfactory memory formation and lamb recognition (0-4 h post-partum) had no effect on ewes maternal behaviour towards their own lambs. It did, however, prevent the normal selective expression of aggressive rejection, and reduced protest vocalisation behaviours directed towards strange lambs. These rejection behaviours did appear 1 h after the termination of tetracaine infusions despite the ewes not being given the opportunity to interact with their own lambs during this time. Therefore, tetracaine blockade of the medial frontal cortex prevents animals from responding with motor aggression, but not vocal aggression, to odour cues from strange lambs, but has no effect on the formation of an olfactory recognition memory for their own lambs. Both pre- and post-partum aggressive rejection of strange lambs was associated with increased concentrations of dopamine, serotonin, glutamate and GABA. When these behaviours were inhibited by the tetracaine infusions, extracellular concentrations of these neurotransmitters were all increased by the anaesthetic but did not change in response to lambs. These findings suggest that a functional medial frontal cortex is not required for the formation of an olfactory recognition memory or for mediating pro-active maternal behaviours. It is however required for the mediation of motor but not vocal aspects of aggressive rejection responses directed towards aversive odour cues from strange lambs.