Maryse Meurisse

Cortical and medial amygdala are both involved in the formation of olfactory offspring memory in sheep

Ewes form a selective olfactory memory for their lambs after 2 h of mother–young interaction following parturition. Once this recognition is established, ewes will subsequently reject any strange lamb approaching the udder (i.e. maternal selectivity). The present study tested the functional contribution of different amygdala nuclei to lamb olfactory memory formation. Using the anaesthetic lidocaine, cortical, medial or basolateral nuclei of the amygdala were transiently inactivated during lamb odour memory formation. Reversible inactivation of either cortical or medial amygdala during the first 8 h postpartum impaired lamb olfactory recognition, whereas inactivation of the basolateral nucleus or infusion of artificial cerebrospinal fluid did not. Control experiments indicate that inactivation of the cortical and medial nuclei of the amygdala specifically disrupt memory formation rather than olfactory perception or memory retrieval. These findings show that both nuclei of the amygdala are required for the formation of a lamb olfactory memory and suggest functional interaction between these two nuclei.

Mapping of brain networks involved in consolidation of lamb recognition memory.

In sheep, ewes at parturition are responsive to any newborn lamb, but within less than 1 h, mothers learn to recognize the odor of their lamb and restrict maternal care to their own offspring (maternal selectivity). In a first experiment, we investigated the long-term retention of maternal selectivity after various mother-young contact and separation durations. After 4 h of contact, 36 h of separation leads to a total loss of selectivity. Increasing contact duration to 7 days prior to this separation maintains selectivity. These data suggest that lamb memory after going through an initial labile state after parturition, is consolidated over time into a more stable long-term memory. Fos immunohistochemistry reveals that reintroduction of the lamb after 4 h of mother-young contact and 3 h of separation activates different maternal brain regions than reintroduction of the lamb after 7 days of mother-young contact and 3 h of separation. While the piriform cortex shows an enhanced activation at both times, a selective enhancement of activation is observed in the frontal medial and orbitofrontal cortices only after 7 days of mother-young contact. These data suggest that as consolidation occurs, the neurobiological networks sustaining lamb memory involve different structures.

Forebrain Structures Specifically Activated by Conditioned Taste Aversion

This study investigates which forebrain structures show Fos protein expression during conditioned taste aversion (CTA) acquisition and whether Fos expression depends on the aversion strength. A novel taste paired with an intraperitoneal injection of a low dose of the malaise-inducing agent lithium chloride (LiCl) induced a weak CTA, whereas associating this novel taste with a high dose of LiCl induced a strong CTA. Increasing the strength of the gastric malaise alone enhanced Fos expression in central, basal, and lateral amygdala nuclei and decreased Fos expression in the nucleus accumbens core. Taste-malaise association induced specific Fos activation in the insular cortex (with both the low and the high doses of LiCl) and the nucleus accumbens shell (with the high LiCl dose only). No significant variation of Fos expression was measured in the perirhinal cortex. Several forebrain areas may be sites of taste-malaise convergence during CTA acquisition depending on the strength of the aversion.

Extensive immunolesions of basal forebrain cholinergic system impair offspring recognition in sheep.

The involvement of the basal forebrain cholinergic system has been extensively investigated in instrumental learning but little is known of its participation in social memory, especially in the memorization of individual traits of a conspecific. The present study tested in sheep its contribution to both instrumental learning and individual offspring recognition. Six weeks before parturition, ewes received injections of a specific cholinergic immunotoxin (ME20.4 IgG-saporin) into the lateral ventricles (150 microg) and in some cases additional immunotoxin injections into the nucleus basalis (11 microg/side). After 3 weeks of recovery, ewes were trained on a classical instrumental visual discrimination task known to be sensitive to cholinergic deficits. The formation of memory of offspring was assessed through both olfactory and visual/auditory recognition tasks. Olfactory recognition was tested by presenting at suckling successively an alien and the familiar lamb at 2 and 4 h after parturition. Visual/auditory recognition of the lamb was performed using a non-olfactory discrimination test between the familiar and an alien lamb after 12 h of mother-young contact. The lesion extent was assessed by counting choline acetyltransferase-immunopositive neurons in the basal forebrain and measuring the density of acetylcholinesterase fibers in different target areas. Results showed that immunotoxic lesions delayed acquisition of the instrumental visual discrimination. Moreover, olfactory recognition of the lamb was severely impaired while visual/auditory lamb recognition was marginally altered. There was no evidence for sensorimotor or motivational deficits. Importantly, impairment was observed in animals for which loss of basal forebrain cholinergic neurons and their efferent fibers was higher than 75%, while striatal cholinergic neurons and Purkinje cells were unaffected. This study provides evidence that the basal forebrain cholinergic system contributes not only to instrumental but also to social learning. In addition, the cholinergic modulation seems of importance for processing visual and olfactory modalities. However, since only extensive lesions affect performance, this indicates that the basal forebrain cholinergic system possesses substantial reserve capacity to sustain cognitive functions.

Distribution and co-localization of choline acetyltransferase and p75 neurotrophin receptors in the sheep basal forebrain: implications for the use of a specific cholinergic immunotoxin

The basal forebrain cholinergic system is involved in different forms of memory. To study its role in social memory in sheep, an immunotoxin, ME20.4 immunoglobulin G (IgG)-saporin, was developed that is specific to basal forebrain cholinergic neurons bearing the p75 neurotrophin receptor. The distribution of sheep cholinergic neurons was mapped with an antibody against choline acetyltransferase. To assess the localization of the p75 receptor on basal forebrain cholinergic neurons, the distribution of p75 receptor-immunoreactive neurons with ME20.4 IgG was examined, and a double-labeling study with antibodies against choline acetyltransferase and p75 receptor was undertaken. The loss of basal forebrain cholinergic neurons and acetylcholinesterase fibers in basal forebrain projection areas was assessed in ewes that had received intracerebroventricular injections of the immunotoxin (50, 100 or 150 microg) alone, as well as, in some of the ewes treated with the highest dose, with bilateral immunotoxin injections in the nucleus basalis (11 microg/side). Results indicated that choline acetyltransferase- and p75 receptor-immunoreactive cells had similar distributions in the medial septum, the vertical and horizontal limbs of the band of Broca, and the nucleus basalis. The double-labeling procedure revealed that 100% of the cholinergic neurons are also p75 receptor positive in the medial septum and in the vertical and horizontal limbs of the band of Broca, and 82% in the nucleus basalis. Moreover, 100% of the p75 receptor-immunoreactive cells of these four nuclei were cholinergic. Combined immunotoxin injections into ventricles and the nucleus basalis produced a near complete loss (80-95%) of basal forebrain cholinergic neurons and acetylcholinesterase-positive fibers in the hippocampus, olfactory bulb and entorhinal cortex. This study provides the first anatomical data concerning the basal forebrain cholinergic system in ungulates. The availability of a selective cholinergic immunotoxin effective in sheep provides a new tool to probe the involvement of basal forebrain cholinergic neurons in cognitive processes in this species.

Afferent and efferent connections of the cortical and medial nuclei of the amygdala in sheep.

The cortical (CoA) and the medial (MeA) nuclei of the amygdala are involved in the processing of olfactory information relevant to social recognition in the ewe. To better understand the neural pathways responsible for these effects, the connections of both CoA and MeA with the telencephalic and diencephalic regions were studied by injecting an anterograde (Biotin-Dextran-Amine, BDA) or a retrograde (Fluorogold, FG) neuronal tracer into either the CoA or the MeA. Concerning the primary olfactory structures, the CoA receives inputs from both the main olfactory bulb and the accessory olfactory bulb (AOB), while the MeA is innervated by cells only from the AOB. Among the other olfactory structures, only the entorhinal cortex and the tenia tecta are connected with both the CoA and the MeA. With respect to the other secondary olfactory structures, the connections with the CoA and the MeA show segregating neuronal routes. The CoA is connected with the accessory olfactory nucleus, the piriform, the endopiriform and the orbitofrontal cortices while the MeA exhibited connections with the nucleus of the lateral olfactory tract, the perirhinal and the insular cortices. Concerning the diencephalic structures, only the MeA receives projections from the PVN and the MBH. On the other hand, we showed that the BNST is the major site of connection with both the CoA and the MeA. Reciprocal projections were observed between the CoA and the MeA and between both nuclei and the basal or the lateral nuclei of the amygdala with the exception of the CoA which does not send inputs to the lateral nucleus. These data are discussed in relation with olfactory learning in the context of sexual and maternal behavior in sheep.

Differential c-Fos expression in the newborn lamb nucleus tractus solitarius and area postrema following ingestion of colostrum or saline

Visceral stimuli and the gut-brain axis play a crucial role in the control of ingestion even in the neonate. The aim of this study was to assess the neuronal activation in the nucleus tractus solitarius (NTS) and the area postrema (AP) following nutritional and non-nutritional stimulations. Lambs received a single gastric infusion of colostrum or saline at 5% birth weight or were sham infused. Infusion of either liquid led to c-Fos-like immunoreactivity (c-FLI) in the NTS and AP. Differences were observed along the sections of the NTS rostro-caudal axis according to the nature of the stimulation, suggesting a specificity of certain afferents and/or NTS areas for nutritional or non-nutritional signals. In the AP, the neuronal activation induced by colostrum was much higher than that induced by saline. A higher number of TH-immunoreactive cells were activated following colostrum infusion, suggesting a specific involvement of the catecholaminergic pathway in the treatment of meal-related stimuli. In spite of functional convergence, the two medullary structures observed responded differently according to the stimulation, indicating a complementary role in the integration of visceral signals.

Behavioral and endocrine evaluation of the stressfulness of single-pen housing compared to group-housing and social isolation conditions

Regulation of neuroendocrine responses is often studied in animals housed indoors in individual contiguous pens. In sheep, these housing conditions are used to control the environment, facilitate biological sampling and limit social stress. However, this type of housing also prevents exploratory behaviors and could induce stereotypies, non-compliant with welfare and possibly associated with a state of stress. In this context, we investigated the impact of housing in a single-pen, with other familiar conspecifics, on emotional state by evaluating behavioral, hormonal and neuronal measures in adult ewes. We hypothesized that emotional state would be more negative in animals housed in a single-pen for one week (Pen) than in freely moving animals (Free) but less negative than in socially isolated subjects (Isol). We tested our hypothesis in ovariectomized ewes to avoid the interaction with sexual steroid variations. Our behavioral, endocrine and neuronal (Fos activation of the corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus) measures confirmed that withdrawing familiar conspecifics was sufficient to induce strong stress responses in Isol ewes, but there was no indication that Pen ewes were stressed. However, the latter showed less mastication activity than Free ewes, probably due to limited accessibility to straw. The highest plasma prolactin levels were observed in Isol and Free animals, which might result from stress and physical activity, respectively. In Free ewes, plasma dopamine was low, consistent with its inhibitory control of prolactin. However, Isol animals had both high levels of prolactin and dopamine, suggesting a dysregulated balance in socially stressed ewes. As in other species, we suggest that the regulation of prolactin by dopamine varies with stress and/or social context. Overall, this study shows that the impact of housing conditions on different neuroendocrine systems should be considered more in the future.

Behavioural and neurobiological effects of colostrum ingestion in the newborn lamb associated with filial bonding.

In sheep, the onset of filial bonding relies on early intake of colostrum. The aim of our work was to describe in the newborn lamb housed with its mother the immediate post‐ingestive effects of colostrum intake, in terms of behaviour and brain activity. In Experiment 1, lambs received five nasogastric infusions of colostrum, or saline, or sham intubations during the first 6 h after birth. Mother–young interactions were recorded before and after the first, third and fifth infusions. The activity of the dam and of the young, which diminished over time in all groups, was temporarily increased in both partners just after each intubation procedure. The number of high‐pitched bleats was significantly lower in lambs that received colostrum than in the sham group, suggesting soothing or satiating properties of colostrum. In Experiment 2, newborn lambs received a single nasogastric infusion of colostrum or saline 4.5 h after birth, or were sham intubated. Neuronal activation was investigated 1.5 h later for maximum c‐Fos activity. Infusion of colostrum and saline induced different patterns of c‐Fos‐like immunoreactivity in the paraventricular and supraoptic nuclei of the hypothalamus as compared with the sham group. A specific oxytocinergic/vasopressinergic (OT/VSP) cell population in the paraventricular nucleus was activated following colostrum and saline infusion, but not sham intubation. Only colostrum induced the activation of the cortical amygdala and insular cortex, two structures involved in learning, associative processes, reward and emotion. We hypothesize that filial bonding may be triggered through colostrum‐rewarded learning/calming processes and that the OT/VSP system may play a role.

Behavioral evidence of heterospecific bonding between the lamb and the human caregiver and mapping of associated brain network

While behavioral mechanisms of bonding between young mammals and humans have been explored, brain structures involved in the establishment of such processes are still unknown. The aim of the study was to identify brain regions activated by the presence of the caregiver. Since human positive interaction plays an important role in the bonding process, activation of specific brain structures by stroking was also examined. Twenty-four female lambs reared in groups of three were fed and stroked daily by a female caregiver between birth and 5-7 weeks of age. At 4 weeks, an isolation-reunion-separation test and a choice test revealed that lambs developed a strong bond with their caregiver. At 5-7 weeks of age, lambs were socially isolated for 90min. They either remained isolated or met their caregiver who stroked them, or not, at regular intervals over a 90-min period. Neuronal activation was investigated at the end of the period for maximum c-Fos expression. Reunion with the caregiver appeased similarly the lambs whether stroking was provided or not. Stroking did not activate a specific brain network compared to no stroking. In both cases, brain regions associated with olfactory, visual and tactile cue processing were activated in the presence of the caregiver, suggesting a multisensory process involved. In addition, activation of the oxytocinergic system in the hypothalamic paraventricular nucleus induced by the presence of the caregiver suggests similar neuroendocrine mechanisms involved in inter-conspecific and animal-human bonding.