Michael R. Hinton

Formation of olfactory memories mediated by nitric oxide.

Sheep learn to recognize the odours of their lambs within two hours of giving birth, and this learning involves synaptic changes within the olfactory bulb,. Specifically, mitral cells become increasingly responsive to the learned odour, which stimulates release of both glutamate and GABA (γ-aminobutyric acid) neurotransmitters from the reciprocal synapses between the excitatory mitral cells and inhibitory granule cells. Nitric oxide (NO) has been implicated in synaptic plasticity in other regions of the brain as a result of its modulation of cyclic GMP levels. Here we investigate the possible role of NO in olfactory learning. We find that the neuronal enzyme nitric oxide synthase (nNOS) is expressed in both mitral and granule cells, whereas the guanylyl cyclase subunits that are required for NO stimulation of cGMP formation are expressed only in mitral cells. Immediately after birth, glutamate levels rise, inducing formation of NO and cGMP, which potentiate glutamate release at the mitral-to-granule cell synapses. Inhibition of nNOS or guanylyl cyclase activity prevents both the potentiation of glutamate release and formation of the olfactory memory. The effects of nNOS inhibition can be reversed by infusion of NO into the olfactory bulb. Once memory has formed, however, inhibition of nNOS or guanylyl cyclase activity cannot impair either its recall or the neurochemical release evoked by the learned lamb odour. Nitric oxide therefore seems to act as a retrograde and/or intracellular messenger, being released from both mitral and granule cells to potentiate glutamate release from mitral cells by modulating cGMP contentrations. We propose that the resulting changes in the functional circuitry of the olfactory bulb underlie the formation of olfactory memories.

Sheep don't forget a face

The human brain has evolved specialized neural mechanisms for visual recognition of faces, which afford us a remarkable ability to discriminate between, remember and think about many hundreds of different individuals. Sheep also recognize and are attracted to individual sheep and humans by their faces, as they possess similar specialized neural systems in the temporal and frontal lobes for assisting in this important social task, including a greater involvement of the right brain hemisphere. Here we show that individual sheep can remember 50 other different sheep faces for over 2 years, and that the specialized neural circuits involved maintain selective encoding of individual sheep and human faces even after long periods of separation.

Oxytocin, amino acid and monoamine release in the region of the medial preoptic area and bed nucleus of the stria terminalis of the sheep during parturition and suckling.

Microdialysis sampling was used to measure the release of oxytocin (OXY) and monoamine and amino acid transmitters from the region of the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (BNST) during parturition and suckling in sheep. Results showed that OXY and gamma-aminobutyric acid release increased in both the MPOA and BNST during parturition and suckling. Noradrenaline (NA) release increased in both structures during parturition but not during suckling. Dopamine (DA) release increased in the MPOA and decreased in the BNST during both parturition and suckling. Aspartate release increased in the MPOA during parturition, and the BNST during suckling, and glutamate release increased in the MPOA and BNST at parturition and only in the BNST during suckling. No changes in the release of serotonin or taurine occurred in these structures during parturition or suckling. In a further experiment on 6 estrogen-primed sheep, OXY (10 micrograms/ml) was infused into the MPOA via bilaterally placed microdialysis probes. This treatment inhibited rejection behavior towards lambs, but did not activate positive maternal responses. These OXY infusions also stimulated release of NA. These results show that complex patterns of neurochemical release occur in two closely related areas of the brain, the BNST and MPOA, during parturition when maternal behavior is stimulated. However, while these patterns of release are similar in the two structures, particularly at birth when maternal behavior is stimulated, they are not identical during labor contractions and suckling. The release of oxytocin within the MPOA during parturition may be important for stimulating a reduction in aggression towards lambs, although this action might be mediated via the effect of OXY on NA release.

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.

Cerebrospinal fluid and plasma concentrations of oxytocin and vasopressin during parturition and vaginocervical stimulation in the sheep

Simultaneous blood and cerebrospinal fluid (CSF) samples were taken from conscious sheep before, during and after parturition. Concentrations of plasma and CSF oxytocin were significantly elevated during contractions and particularly at birth. Mean prepartum CSF concentrations of oxytocin were around 55% of those found in plasma but postpartum they were up to 2-fold higher than those in plasma. Plasma concentrations of oxytocin were only significantly elevated, compared to prepartum levels, for 15 min postpartum whereas those in CSF were increased for the whole of the 120 min postpartum sampling period. Plasma, but not CSF, concentrations of arginine-vasopressin (AVP) were significantly raised during contractions and birth, and for 15 min postpartum. During the prepartum period CSF AVP concentrations were 67% of those found in plasma whereas at birth plasma levels were 10-fold higher than in CSF. In a separate experiment it was shown that 5 min of mechanical vaginocervical stimulation also stimulated significant increases in CSF and plasma oxytocin concentrations and in plasma vasopressin. Results support previous work suggesting an important role for central oxytocin release in the postpartum induction of maternal behavior and demonstrate that elevated concentrations of oxytocin in the CSF are present for a greater period than in blood. Elevated plasma AVP concentrations during contractions, birth or vaginocervical stimulation may be stimulated by stress associated with these stimuli.

Are faces special for sheep ? Evidence from facial and object discrimination learning tests showing effects of inversion and social familiarity

We have previously shown that sheep, like monkeys, have neural circuits within the temporal lobe that respond preferentially to faces. They can also discriminate between sheep, humans and other animals on the basis of facial cues using an enclosed Y-maze. In the present study we investigated the speed with which Clun Forest sheep learn to discriminate between familiar and unfamiliar faces, as opposed to symbols, in order to gain a food reward using the same Y-maze apparatus. Animals (n = 10) received 1 day of training where projected images of the pairs of faces or symbols were paired for 20 trials with a picture of either an empty or full bucket of food (which indicated which choice of face or symbol would result in the animal receiving a food reward) and on the next 4 days they were given a further 20 trials a day with the faces or symbols alone. Results showed that sheep learned significantly faster (by day 1 or 2 post training) to recognise sheep faces of a familiar breed compared to geometrical symbols (3-4 days post training). Learning using faces of animals of another unfamiliar breed was also significantly better than for symbols but was significantly worse than that seen using faces of a familiar breed. Inverting the faces significantly reduced learning speed for faces of a familiar breed but not for that of an unfamiliar one. Inverting familiar objects, food buckets, also did not impair discriminatory performance. In a further set of trials where discrimination learning was made more difficult by excluding cued trials and reducing the number of daily trials to eight, social familiarity was found to further improve the animals ability to learn to discriminate between the faces of a familiar breed. Finally, while discriminatory performance for adult sheep faces was very good, that for young lamb faces was poor, with only one animal learning to choose the face associated with food. It was confirmed in maternal ewes that they were also slow to learn to recognise the faces of their lambs (2-3 weeks). Overall these results show that sheep can learn to distinguish between individual adult sheep faces but that breed and social familiarity influence the level of performance. Further, discrimination learning of familiar and unfamiliar facial stimuli is better than between simple geometrical symbols, indicating that faces may be preferentially processed by the brain compared to other objects suggesting that faces are indeed special in this species as has been claimed for human and non-human primates.

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.

Microdialysis measurement of neurochemical changes in the mediobasal hypothalamus of ovariectomized ewes during oestrus

Oestrus behaviour and the luteinizing hormone (LH) surge are induced in ovariectomized ewes by oestradiol (E2) after a period of progesterone priming with a low level of E2 (Pge2) and we have previously shown that these effects are primarily mediated through their action on the mediobasal hypothalamus (MBH). The aim of the present study was to assess what neurochemical changes in the MBH are induced by these steroids that might mediate their action on oestrus behaviour and LH release. Eight ovariectomized ewes were implanted with microdialysis probes in the MBH and submitted to three artificial cycles, so that they exhibited either both oestrus behaviour and an LH surge (Pge2 + E2), an LH surge alone (E2 alone) or neither oestrus behaviour nor an LH surge (Pge2 alone). Microdialysis and blood samples were collected every 30 min from 4 h before the end of Pge2 treatment until the end of oestrus. Behavioural tests with a ram were made to assess receptivity. Dopamine (DA) levels were found to increase significantly at the termination of Pge2 treatment after both Pge2 + E2 and Pge2 treatments. When the ewes received E2 after a Pge2 + low estradiol priming (Pge2 + E2), DA levels decreased 16 h later (4 h after E2) whereas they did not change after E2 or Pge2 alone. By contrast, serotonin (5HT) levels did not change significantly during the first 24 h but then increased when ewes received E2 alone and decreased when they were treated with Pge2 + E2. gamma-Aminobutyric acid (GABA) concentrations decreased significantly at the beginning of the sampling period after all treatments but this decrease lasted longer after Pge2 + E2 and was most pronounced at the beginning of receptivity. No significant long term effects of these steroid treatments were found on noradrenaline (NA), aspartate, glutamate, glycine and taurine levels. However, E2 administration was followed during the next few hours by a significant increase in glycine and to a smaller extent in glutamate and GABA. More importantly, when ewes were treated with Pge2 + E2, NA levels increased significantly following the behavioural interactions with a ram when the ewes were sexually receptive. In contrast to this, DA levels only increased during interactions with the ram when the ewes were not receptive. 5HT levels increased after tests where the ewe was either receptive or unreceptive to the male. GABA, aspartate and glycine levels increased in the sample just preceding the test and then decreased during it.(ABSTRACT TRUNCATED AT 400 WORDS)

A simple method for fostering lambs using anoestrous ewes with artificially induced lactation and maternal behaviour

Abstract The possibility of a simple method for using anoestrous ewes to foster orphan or triplet lambs was investigated. Twenty-two anoestrous ewes (12 multiparous and ten nulliparous) were given vaginal sponges containing medroxyprogesterone acetate (250 mg) and oestradiol dipropionate (ODP) (25 mg) for 2 weeks. When these were removed a second sponge was inserted containing the same concentration of progesterone and 50 mg of ODP. This second sponge was left in place for a further 4 weeks and significant udder development (one-quarter to one-third of that seen in animals at parturition) was seen in all of the multiparous ewes and in eight out of ten of the nulliparous ones. Plasma prolactin concentrations in these animals were increased around ten-fold by this treatment and the first milk expressed after the 6 week treatment period had the colouring and consistency of colostrum. Twelve of the ewes (eight multiparous and four nulliparous) were presented with lambs ranging from 30 min to 6 days old. None of the ewes showed full maternal acceptance of lambs following the hormone treatment alone, but after 2–3 min of mechanical stimulation of the vagina and cervix they all immediately became maternal towards the lambs, formed a selective bond with them within 3 h, and reared them successfully through until weaning (one lamb died 1 week after fostering). The initial quality of maternal care was indistinguishable from that of 12 normal post-partum ewes. Only one lamb was raised by each ewe due to the low level of lactation induced, but in all cases udder development was increased as a result of suckling and by 14–16 weeks post-fostering there were no obvious differences between the sizes of lambs reared by normal post-parturient sheep and those reared by the artificial foster mothers.

Biphasic role of dopamine on female sexual behaviour via D2 receptors in the mediobasal hypothalamus.

Dopamine has been implicated in the control of sexual behaviour, but its role seems quite complex and controversial. The aim of the present experiments was to investigate the effects of dopamine (DA) acting on D2 receptors in the mediobasal hypothalamus (MBH) on sexual behaviour in female sheep. To achieve this, the D2 agonist, quinpirole, was administered bilaterally via microdialysis probes into the MBH of ovariectomized ewes either before or after oestradiol (E2) administration. Quinpirole (100 ng/ml) infused for 6 h just before E2 hastened the onset of oestrus behaviour and the luteinizing hormone surge, whereas the same treatment given 6-12 h or 18-21 h after E2 decreased the intensity of sexual receptivity without affecting LH or prolactin secretion. We then tested the hypothesis that E2 stimulates the onset of oestrus partly by decreasing DA activation of D2 receptors. In this case the D2 antagonists pimozide or spiperone (100 ng/ml) were infused into the MBH via microdialysis probes for 11 h in the absence of E2 administration. A significant number of ewes showed induction of receptivity with both antagonists, although its intensity was significantly lower than that induced by E2. These treatments generally did not significantly alter extracellular concentrations of monoamines or aminoacids although quinpirole modulated the ability of sexual interactions to increase noradrenaline release. These experiments show that DA acts via D2 receptors in the MBH to control female sexual behaviour in a biphasic manner: the onset of sexual motivation and receptivity requiring an initial increase in activation followed by a decrease. This dual action could explain some of the controversies concerning DA action on sexual behaviour.