Mammary olfactory signalisation in females and odor processing in neonates: Ways evolved by rabbits and humans

Behavioural Brain Research 200 (2009) 346–358

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Mammary olfactory signalisation in females and odor processing in neonates: Ways evolved by rabbits and humans Benoist Schaal ∗ , Gérard Coureaud, Sébastien Doucet, Maryse Delaunay-El Allam, Anne-Sophie Moncomble, Delphine Montigny, Bruno Patris, André Holley Centre Européen des Sciences du Goût, Groupe d’Ethologie et de Psychobiologie Sensorielle, CNRS, Université de Bourgogne, 21000 Dijon, France

a r t i c l e

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Article history: Received 2 December 2008 Received in revised form 9 February 2009 Accepted 9 February 2009 Available online 14 February 2009 Keywords: Olfaction Pheromone Mother–infant relations Neonate Milk Rabbit (Oryctolagus cuniculus) Human

a b s t r a c t Mammalian females have long been known to release olfactory attraction in their offspring. Mammary odor cues control infant state, attention and directional responses, delay distress responses, stimulate breathing and positive oral actions, and finally can boost learning. Here, we survey female–offspring odor communication in two mammalian species – European rabbits and humans – taken as representatives of evolutionary extremes in terms of structure and dynamics of mother–infant relations, and level of neonatal autonomy. Despite these early psychobiological differences, females in both species have evolved mammary structures combining multiple sources of endogenous and exogenous odorants, and of greasy fixatives, conferring on them a chemocommunicative function. To process these mammary chemosignals, neonates have co-evolved multiple perceptual mechanisms. Their behaviour appears to be driven by plastic mechanism(s) calibrated by circumstantial odor experience in preceding and current environments (fetal and postnatal induction of sensory processes and learning), and by predisposed mechanisms supported by pathways that may be hard-wired to detect species-specific signals. In rabbit neonates, predisposed and plastic mechanisms are working inclusively. In human neonates, only plastic mechanisms could be demonstrated so far. These mammary signals and cues confer success in offspring’s approach and exploration of maternal body surface, and ensuing effective initial feeds and rapid learning of maternal identity. Although the duration of the impact of these mammary signals is variable in newborns of species exposed to contrasting life-history patterns, their functional role in setting on infant–mother interaction in the context of milk transfer can be crucial. © 2009 Elsevier B.V. All rights reserved.

1. Introduction All mammalian offsprings have to surpass major life-history transitions in early development. Right after birth, they must succeed in orienting towards the female, in reaching the source of milk, in taking in a sufficient dose of milk, and in efficiently processing it both into biomass and into useful sensory information. Later in life, they have to more or less progressively shift from the exclusive milk diet to the local non-milk diet of adult conspecifics. These perinatal and weaning transition periods culminate in selective pressure on the adaptive abilities that mammalian offsprings have to mobilize. These selective constraints on neonatal adaptation go hand in hand with the evolutionary elaboration of strategies in the mother-to-infant transmission of information, in the rearing of infants who can sense and learn this information, and in the

∗ Corresponding author at: Centre Européen des Sciences du Goût, CNRS (UMR 5170), Université, de Bourgogne-Inr a, 15 rue Picardet, 21000 Dijon, France. Tel.: +33 3 80 68 16 00. E-mail address: [email protected] (B. Schaal). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.02.008

ways these information exchanges are synchronized and regulated within the functional entity constituted by the female–offspring dyad. The milk transfer/acquisition period will be taken here as a paradigmatic context to analyze commonalities of information encoding/decoding strategies in mammalian mother–infant dyads. The emphasis will be here on nasal chemoreception, which represents a highly conserved modality in newborn organisms throughout the mammalian radiation. Some mammals give indeed birth to neonates that are highly immature and dependent in terms of physiological autonomy and sensory, cognitive, and motor abilities (e.g., marsupials, altricial rodents or carnivores), newborns in other species being highly mature on sensory criteria, although their level of motor maturation can be variable (e.g., precocial sensory, physiological and motor functionality in ungulate or some rodent neonates; vs. sensorily mature, but motorically altricial primate infants). But regardless of such disparities in neonatal sensory and response capabilities, olfaction is universally well developed in mammalian newborns to deal with odor stimuli that are unavoidably emitted by, or associated with, the maternal body. To illustrate our point, we will consider here two mammalian cases, European rabbits (Oryctolagus cuniculus) and humans (Homo

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Table 1 Some maternal and neonatal features related to breeding biology, lactational physiology, milk composition, nursing and sucking behaviour in two species representing extreme strategies of mother–infant relationships in mammals, the European rabbit (Oryctolagus cuniculus) and the human (Homo sapiens). Oryctolagus

Homo

Generala Ecological and trophic position Breeding niche construction Parenting (and antipredator) strategy Mean n offspring/gestation Mean n nipples

Herbivory/prey Nidicoly (breeding burrow + fur + vegetal nest) ‘Absentee’: (protection by absence) Wild: 4–6/domestic: 4–15 6–8

Omnivory/predator Matricoly (on mother or on helpers) ‘Continuous’: (protection by presence) 1–2 2

Maternal behaviour–lactation–nursingb Duration of lactation time budget with offspring -during early lactation Inter-feed intervals Duration/Frequency of feeds Arousal state while suckling Mother-to-young bonding

∼1 month ∼0.01% (postpartum weeks 1–2) widely spaced 5 min/24 h Hyper-aroused ??

1–36 monthsc 100%c (postpartum weeks 1–4) Frequentc 10–20 min/1 hc Generally relaxed Strong affective links

Milkd Composition -% fat/proteins/sugars

15.2/10.3/1.5

3.5/1.2/7

Neonatee Sensory functional onset -Somesthesis -Chemoreception (olfaction, taste) -Audition -Vision

Birth Birth Day 7 Days 10–11

Birth Birth Birth Birth

Neonatal behaviour/physiology -Sucking dynamics -Intake %/body weight -Gastric emptying -Thermal regulation

Fast suckers 25–35%/5 min Slow Immature

Slow suckers 2–3%/20 min Fast Immature

Young-to-mother attachment

??

Strong affective links

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Sources in [47,66,77]. Sources in [77,82,112,167]. These criteria can be exceedingly variable across cultures. Sources in [22,37,70,78]. Sources in [46,50,82,163,167].

sapiens), taken as representatives of evolutionary extremes in terms of reproductive strategies, social structure of mother–infant relations (viz., exclusivity, quality, and duration of interactions), level of neonatal autonomy, and pace of development during and beyond the lactation period (cf. Table 1). The females of both species will be considered as committed in maternal effects, i.e., the transmission of information that rests on the environment that females create around their offsprings during fetal and postnatal development. Emphasizing olfaction in that background, our goal is to attempt to better grasp some mechanisms by which mammalian females shape the sensory ecology of their progeny, and at the same time fine-tune their early neuro-sensory, cognitive and behavioural development. 2. Selective constraints on early developmental transitions Newly born mammals are exposed to an extremely sturdy selective pressure leading to mortality figures that emphasize the costs of maladaptive responses in the female–offspring dyad. Two early mammalian transitions—perinatal and weaning—concentrate peak challenges in terms of physiological, behavioural, cognitive and social changes, and hence delimit periods of increased threats to individual viability of young mammals. Here we will consider only the birth transition and the critical need of rabbit and human newborns for the timely-viz. as quick as possible after birth-ingestion of colostrum and milk. Neonatal rabbits have to pass a particularly tight adaptive bottleneck to reach the milk resource in terms of physical availability of the female, of nipple-focused sensory-motor competence and of competition among littermates. During the first 2 weeks post-

partum, they have to find and suck nipples within the 3–5 min the female makes herself accessible over the day [82,144,167]. The number of littermates (6–15) aiming at milk often exceeds affordable nipples (8–10, depending on domestic breeds), inducing intense sibling rivalry [44]. But as individual pups do not monopolize a nipple over a same nursing bout [6,43], in principle all valid pups can suck. Thus, despite strongly restrictive conditions of nipple access, the sucking of individual pups is generally possible, its success being further warranted by the fine-tuning of motor skills that improve from day to day through maturation and learning [61,109]. In addition, any gain in body weight is advantageous in the competition between littermates [7,26,44]. In humans as well, neonatal breastfeeding initiation is a period of acute threat to viability. The ability to initiate sucking is highly variable among individual newborns. Hence, postnatal delays in establishing optimal colostrum/milk transfer from mother to infant are far from uncommon. For example, a recent Californian study revealed that 49% and 22% of normal term-born infants express non-optimal breastfeeding on the day of birth and on day 3, respectively; these unfavourable outcomes were strongly associated with maternal inexperience (i.e., primiparity) [39,96]. Such inadequate milk transfer during the first feeds, if not handled rapidly, can lead to excessive weight loss, dehydration, and serious threat to viability [23,111]. Further, in conditions presumed to bear some resemblance with those that prevailed during human evolutionary history, a study following up home births in today’s rural Ghana showed that a 1-day postponement in the initiation of breastfeeding explained 16% of neonatal losses. Moreover, a delay of only 1 h postbirth to engage breastfeeding explained 22% of neonatal mortality [45].

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In both species under scrutiny, there is thus an exceedingly strong selective pressure for females to make their mammae accessible and for neonates to reach them as quickly as possible after birth for effective colostrum transfer and intake. Consequently, any sensory, behavioural and cognitive means in newly born organisms that can speed up this localisatory task and acceptance of the nipple should have had beneficial effects on neonatal viability and adaptive life onset over evolutionary time. The mammary structures, the only structures of the mammalian maternal body which infants have to obligatorily contact to survive, are thus expected to carry cues and signals capable of prompting adequate responsiveness in “naïve” newborns confronted to suckling for the very first times. 3. The first task of mammalian mothers: making the milk source conspicuous In both species under consideration, natal body weight is highly protective towards episodes of inanition and associated risks in neonates [21]. It depends first on gestational conditions (placental function; maternal nutrition and exposure to stress; and, in polytocous species, fetal position within the uterus; e.g., Ref. [125]). Afterwards, during the neonatal period, it depends on the mother’s lactational performance and the newborn’s realisation of efficient strategies to acquire and convert milk into biological tissues, based on social contact (with mother, littermates, or both) to keep warm and immune from costly stress responses due to separation. It also depends on the ability of the female to advertise the mammary region, in presenting herself adequately in time and space to their offspring, and promoting them to trace cues beaconing a nipple. In general, mammalian newborns initially exhibit lengthy and strenuous searching on (or under) the mother’s pectoral or abdominal areas, before localising the mammary area, seizing a nipple and finally sucking it to extract colostrum. Even human infants, who are fully assisted in finding the breast, show considerable interindividual variability in the latency to latch on a nipple [120] and in optimal milk intake over the first postnatal days [39]. The rapid intake of colostrum being critical to engage energetic and hydric metabolism, passive immunity and learning in offspring, a strong selective pressure has driven mammalian females to evolve devices – nipples or teats – that are easily localisable and orally seizable by inexperienced newborns. Among the species studied so far (e.g., rat, rabbit, cat, dog, sheep, pig, human) [1,12,13,95,114,123,124,140], it appears that the most conserved strategy to increase the conspicuousness of the nipples relies on perioral touch and olfaction. Regardless of the newborn’s position in the altricial–precocial continuum and of the species-specific pattern of nursing behaviour, mammalian females emit some sort of chemical signals in, on or around their mammae, and their infants are born with all useful sensory and cognitive means to process these signals, as well as with apt motor resources to appropriately react to them. The sensory salience of olfaction in this context is highlighted by experiments impeding neonatal organisms’ ability to process odors. Rabbit newborns rendered anosmic by bulbectomy or peripheral inactivation of the olfactory mucosa (ZnSO4 flush) loose their ability to locate a nipple and consequently starve to death if they are not hand-raised [142,143]. A less drastic impediment to the olfactory mediation of nipple finding has consisted in altering the signal or the source of active cues. If the normal abdominal odor cues of a lactating rabbit are washed off, the pups are delayed in finding nipples [109]. Further, covering nipples with a rubber film has the effect of disrupting normal nipple searching behaviour, at various rates however depending on the extent and location of the hindrance created on the nipple surface [27,61]. Similarly, when the breast of a lactating woman is altered by intrusive washing or by more gentle chemo-emissive disruption,

human infants display alterations in their behaviour. In the first case, they appear to take more time and to be less successful in reaching the washed breast as compared to the olfactorily intact breast [157]. In the second case, when the breast is merely covered with a scentless transparent plastic film, newborns held to the breast display less attraction and mouthing responses, less visual attention, and more rapid release of crying [40]. Thus, Oryctolagus as well as Homo mothers emit over their mammary surface odor cues that clearly affect their offspring behaviour. These mammary odor cues appear to be specifically emitted by lactating females, suggesting physiologically primed variations in terms of either qualitative properties or level of emission above a certain threshold. Female rabbits are particularly potent in releasing head searching and oral grasping in pups exposed to their abdominal fur. When put in direct contact with [62,64] or above the abdomen of rabbits [24], pups do respond with more intense orientation and oral grasping to the abdominal odor of lactating females relative to the same odor from non-lactating females. Taking advantage of a meat-production rabitry, Moncomble et al. [99] exposed pups to excised nipples from lactating and non-lactating females, hence excluding sources of odor cues outside the nipple surface. While nipples from lactating females were highly potent in eliciting the activation of searching and oral grasping responses in pups, the nipples from non-lactating females were completely inactive. Further, in the lactating females, the differential behavioural activity was restricted to the nipples, adjacent skin patches and fur being ineffective. The nipples of lactating rabbits can thus be seen as islets of odor cues that are particularly powerful to pups in the inactive background of surrounding fur. Converging, although less complete, data are at hand in the human case. When neonates are exposed to the odor of cotton pads worn on the breast by either a postparturient lactating woman or by a non-parturient woman, they orient more to the former than to the latter [117]. Although infants can discriminate their mother’s from another mother’s milk [85], such individual-specific cues were not involved here. The above tests were indeed run in exposing exclusively bottle-fed infants to the breast odor of lactating women who were unfamiliar and unrelated to them, indicating that the attractive potency of such odors does not seem to derive from postnatal learning. Thus, as far as both model species under consideration allow to generalize, some efficient volatile factors appear to be linked with the physiological state of lactation in mammalian females, and even more (at least in the rabbit) with the stage of lactation and the moment of odor sampling relative to nursing (cf. Section 6). Whether the test female is the mother or an unfamiliar female does not seem to matter, indicating that lactating females emit invariable mammary signal(s) the attractive power of which overrides individual-specific cues. 4. Mammary odor sources By which means are nipples made olfactorily conspicuous to the naïve neonates of both species under consideration? Contributing sources are obviously complex, involving multiple surface cues and internal cues. The former emanate from secretory productions or deposits, potentially reworked by enzymes from infant saliva or the local skin flora. Internal cues are carried in colostrum/milk in the form of odorous metabolites reflecting mother’s diet, and possibly also her psychological and physiological state, and genetic and immunogenetic constitution. In rabbit females, the active compound(s) comes from the nipples, but little is known about the precise histological sites releasing them. The nipple epidermis being more reactogenic to newborns than the surrounding skin or fur [99], all analyses were directed to the nipple itself. Increased keratinisation on the whole nipple

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surface of lactating females [98] supports higher release of lipids derived from epidermal apoptosis [2]. Well-developed sebaceous glands are located only at the lower, proximal part of the nipple [98]. The sebum emitted from this basal ring of glands may thus be involved in facilitating the sucking-seal between the pups’ lips and nipple, but also in some signalling function by themselves or by their ability to sequester some active milk compounds. Washing away these surface secretions from excised nipples with organic solvents has the effect to reduce or completely abolish their potency in releasing oral grasping in newborn pups. Finally, skin lipids from this basal ring of sebaceous glands may also create a barrier effect limiting the local spreading of milk-related compounds beyond the surface of the nipple into the surrounding fur, hence restricting the active cues to the nipples themselves. Otherwise, the internal sources of compounds potentially involved in the nipples’ attraction to rabbit pups are obviously carried in milk. It may be noted that fresh rabbit milk itself is powerful in releasing the pups’ searching–grasping response [29,73,109,138]. The behavioural activity of rabbit milk odor is time-dependent, however, fading to complete abolition after a 1.5 h-rest in ambient air [73]. But, interestingly enough, the odor of the surface of excised nipples from lactating rabbit females does not fade away with passing time [98], suggesting that some surface compounds (epidermal lipids, sebum) bear either intrinsic behavioural activity or do preserve the activity of some trace remnants of milk left on the nipple surface after nursing. The compounds rendering rabbit milk behaviourally active to neonates appear to originate either from environmental sources transferred into milk or from de novo secretion processes along the mammary tract. Environmental influences on milk cues will be discussed below. The intra-mammary source of active compounds is attested by an experiment that compared the activity of milk samples collected either from the alveolae, from the duct below the nipple level, or right after ejection [99]. It came out that only ejected milk was efficient, designating the terminal part of the milk ducts as the possible source of active compounds. Histological analyses revealed indeed that, in lactating rabbits, the milk ducts are enlarged at the nipple level in the form of a sinus which wall is extremely convoluted and lined with secretory epithelium [98]. This tortuous, elaborate structure suggests an exchange surface optimizing the release of ductal secretions into the passing milk flow. Alternatively, some involatile precursors carried in milk may be instantaneously oxidised when contacting air, releasing volatile compounds. The human females’ mammary structure obviously differs from that of the rabbit. But beyond morphological variations (number and size of nipples, and their surrounding glabrous skin demarcating the areola), the human nipple/areolar region is similarly supplied with all types of skin glands. The nipple itself abounds in apocrine and sebaceous glands with ducts opening on its tip and giving off secretions during lactation [101,116]. On the areolae, eccrine sweat glands and large sebaceous glands can be found [101]. Additionally, the areolar surface is speckled with small prominences, named Montgomery’s tubercles or glands [102]. These areolar glands (AG) of Montgomery result from the coalescence of sebaceous glands and miniature mammary acini [100,148]. A quantitative assessment of AG prevalence, distribution and activity was run in two samples totalising 121 postparturient women engaged in breastfeeding [42,139]. The majority of subjects (97%) bore more than 1 AG, and 83% bore from 1 to 20 AG per areola. These AG can give off a visible lactescent fluid. This oozing of areolar fluid was confirmed in about 1 out of 5 women, in which at least one AG secreted between nursing bouts (this proportion may be underestimated, however, as the AG census was made amid two nursing bouts without distal (crying) or proximal (suckling) infant stimuli known to trigger milk release). This polymorphism in

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AG number/activity may be balanced in part by their non-random distribution on the areolae. The AG appear indeed unevenly distributed over the areolae [42,139], more of them (particularly, the secretory AG) scattering the upper and lateral areolar quadrants. This repartition bias on zones towards which the infants’ noses are mostly directed during nursing may favour their communicative agency. Colostrum and milk released at the nipples from the main lactiferous ducts massively add their intrinsic olfactory qualities to the areolae. The most obvious (although insufficiently documented) influence comes from odorigenic compounds transferred from maternal diet. Empirical evidence that aromas from various foods (e.g., garlic, vanilla, alcohol, carrot) or experimental odorants administered in capsules easily pass into milk has been obtained by regularly sampling milk after human mothers ingested them [55,90–92,94,130]. Subsequent odor changes of milk, culminating in intensity 2–3 h after ingestion, are detectable to nurslings who modify their sucking pattern and milk consumption. Further, maternally inhaled odorants (e.g., combusting tobacco) are also transferred into milk, where they can easily be sniffed out by adult noses [93]. Finally, maternal exercise-induced changes in milk composition can affect milk composition and be detected by nurslings [161]. Together these varied sources of substrates may create a complex odor cocktail. The lipid fraction from keratinizing epidermis, sebum originating from free sebaceous glands and from AG, as well as fatty acids from milk, may act as odor fixatives that improve the chemical and temporal stability of the olfactory complex formed on the areolae. The intricate arrangement of sebaceous and lacteal sources within the AG [148] may indeed favour the mingling of sebum and areolar milk during sucking episodes. However, local biochemical or physical processes may act on the selective release of given compounds or categories of compounds of the mixture. For example, salivary enzymes deposited by the suckling infant may influence a differential liberation of odor-active compounds [19]. Fluctuations in skin temperature may also differentially affect the evaporation rate of volatiles composing the local mixture (see below). In summary, the nipple (in both species) and surrounding areola (in humans), combining multiple sources of endogenous and exogenous odorants, of greasy fixatives, of enzymes, as well as a heat-based diffusion device (cf. below), may be ascribed a chemocommunicative function in the young organism’s guidance to the source of milk. 5. Deconstructing complex systems of mammary cues and signals From the above evidence for variety and intricacy of sources of behaviourally active odor substrates, the chemistry that mammalian females present to their offspring is expected to be commensurably complex. A first partition in this complexity is between volatile and involatile fractions of the mixture of mammary cues, leading either to detection from a distance or to the need for direct contact for perception to occur. Involatile proteins, lipids or hydrocarbon can in this way act as fixatives or precursors of volatile cues, as well as ready-to-work chemosignals. A second divide in the complexity of the mammary odor mixture is between individual-specific and species-specific compounds. Some compounds reflect indeed idiosyncratic characteristics of the female (e.g., her diet, level of stress, physiological state), others representing higher level categories of meanings (kin categories, population or species/genus). Depending on the behavioural tests used to assess responses, an organisms’ ability to extract individual or supra-individual meanings can be evidenced in a same chemosignal. Thus, complex social chemosignals aggregate multiple levels of

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information that can be perceptually segmented by neonatal brains [110,131,164]. 5.1. Individual-specific vs. species-specific cues in milk In the present context, species-specificity of odor cues can be characterized in different ways: either by presenting ‘naïve’ newborns with given cues from unrelated female conspecifics living in different environments, by exposing them to homologous compounds from heterospecific females, or conversely by presenting the target homospecific substrate to newborns of other species. From such experiments, it comes out that rabbit pups can simultaneously attend to several types of odor cues carried in rabbit milk. They display a greater preference for individual-specific qualities of milk, such as when it carries odors that were already experienced in utero. For example, pups born to cumin-fed females seize more readily a glass-stick carrying milk from cumin-fed females, while conversely pups born to females fed standard diet more readily grasp a stick dipped in the milk from females receiving that same diet [29]. However, these pups also respond at a much higher rate to any sample of rabbit milk than to the control stimulus (water), indicating that rabbit milk releases oral grasping regardless of its individual-specific aroma content. Thus, neonatal rabbits can differentiate odorants that are unique to their developmental ecology from odorants that are common to all rabbits. Such experiments made clear that female rabbits and their milk are particularly potent in releasing oral grasping in pups. Rabbit pups do not respond in such a way when put on anaesthetized lactating rats, cats, or hares [59,109]. But when put in contact with or above (without contact with) the abdomen of rabbit females, they do respond regardless of whether the test animal is the mother or an unfamiliar female [24,27,62,109]. Thus, lactating rabbits emit species-specific cues which attractive power on pups clearly overrides individualspecific cues. Further, the abdominal odor of lactating females is as attractive as the odor of fresh rabbit milk when opposed in a choice test [27], telling that particularly active cues are emitted in, on or around the nipple based on compounds that may be common to milk and surface cues. In assaying rabbit, bovine, ovine, porcine and feline milks on rabbit pups, Müller [109] evidenced that rabbit milk odor is exclusive in eliciting the responses of newborn rabbits. He further found that the releasing potency of rabbit milk is rapidly lost after milking (see also [27,74,138]). Thus, all evidence converged to designate rabbit milk as the vehicle of a potent odorous factor eliciting orientation, rooting and nipple attachment in rabbit pups. Similar results prevail in human newborns that exhibit a preference for any human milk odor over non-specific milk (e.g., cow milk-based formula). When both types of milk are presented in a paired-choice test to 4 day-old infants that never directly experienced human milk (bottle-fed since birth), human milk odor elicits equal attraction (head-turning) and higher appetence (oral activity) than the odor of the formula engaged in satiation for about 25 times since birth [87]. A converging result was reached in comparing the response of breastfed newborns’ to an extraneous odorant (camomile) applied on the mother’s breast and to the odor of the mother’s milk [38]: both stimuli elicited equal attraction indicating that the olfactory attractiveness of conspecific milk cannot be easily surpassed by an arbitrary odorant that has been associatively engaged with the multiple reinforcing potency of breastfeeding. 5.2. A species-specific chemosignal in rabbit milk Milk is physically, chemically, and biochemically multifaceted [54,113]. Which fraction(s) of it bear(s) chemosensorily induced behavioural activity to rabbit and human newborns? A first step in

reducing this huge complexity is to limit the analysis to the volatile compounds collecting in the headspace that develops over standing milk. But even then the profile of volatiles from milk remains exquisitely complex, leading to gas chromatographic (GC) tracings composed of more than 150 peaks in the case of rabbit milk [138], or mixture containing more than 40 odor-active compounds in human milk (e.g., Refs. [20,47]). A further step consists in pinpointing GC peaks which bear significant behavioural impact. In applying a GC-olfaction assay using rabbit pups as “noses”, target milk volatiles could be simultaneously detected by the biological sensor and by the physical sensor of the chromatograph [138]. A reactogenic pattern of 21 peaks was thus identified by GC-mass spectrometry, the corresponding compounds being subsequently presented as highly diluted, pure stimuli to neonate pups. Among these 21 candidate compounds, a single one, 2-methyl-but-2-enal (2MB2), was found to be effective in >90% of pups [28,30,138]. The key role of 2MB2 in the odor of rabbit milk was further corroborated in relating it to the above-reported decrease in behavioural activity of milk left standing. The decrease of milk activity correlated indeed with the fall in 2MB2 concentrations, indicating the clear parallel of 2MB2 content in the milk headspace and the releasing potency of milk. In addition, when behaviourally inactivated rabbit milk was spiked with 2MB2 in adequate concentrations, its full behavioural effect was reinstated [138]. 5.3. A pheromone in rabbit milk The adequacy of entering 2MB2 into the class of biologically active substances named ‘pheromones’ was for us conditional upon verifying the currently most stringent definition of the concept as it applies to mammals [9]. These criteria imply that the candidate compound(s) is (1) chemically simple (one or a small set of compounds in fixed ratio), (2) release(s) unambiguous, morphologically invariant, and functionally obvious behavioural or physiological responses in a receiver (3) in a highly selective and (4) speciesspecific manner; finally, (5) the stimulus-response coupling should not depend on previous sensory experience with the stimulus. It came out that 2MB2 is a monomolecular signal eliciting responses which structure and frequency are similar to that triggered by the original mixture (milk), regardless of the mode or context of presentation. The selective activity of the compound was ascertained not to be due to novelty or non-specific arousal effects by comparing the rate of pup responsiveness elicited by 20 reference odorants that are (or not) present in rabbit milk [30]. The generality at the genus/species level of the releasing potency of 2MB2 was positively established in testing either pups from different breeds, pups from different colonies exposed to varied diets, or wild-type rabbit pups [35,138]. In addition, presenting 2MB2 to neonatal rats, mice, cats, and even closely related brown hares (Lepus europaeus), came out with negative results [138]. Finally, the degree of independence of 2MB2’s activity from exposure effects was verified in assessing whether it could be induced by prenatal acquisition, by facilitated learning during the natal process, or by rapid learning immediately after birth. Postnatal or natal learning was ruled out in testing pups deprived of 2MB2 exposure (i.e., separated from the mother after caesarean delivery). Further, the facts that amniotic fluid and pregnant female’s blood were behaviourally ineffective, and that 2MB2 could not be traced in these substrates by GC-MS [138], suggests that its activity may not derive from prenatal induction. It was only after we obtained the above set of results verifying the criteria proposed by Beauchamp et al. [9] and Johnston [72] that we dared qualifying 2MB2 from rabbit milk as a pheromone. As 2MB2 appears to be produced somewhere in the mammary tract and to be emitted in milk, it was named “Mammary Pheromone” (MP).

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So far, no evidence for a similarly well-identified volatile compound intervening in mother–infant interactions is at hand in our own species. However, unidentified compounds carried in human milk and/or in areolar secretions have been shown to indisputably release appetitive responses in human newborns [83,87,127,132,158]. These substrates now await further physicochemical or chromatographic fractionation of mammary substrates, identification of their molecular constituents, and subsequent systematic screening of candidate compounds by the nose of infants [41,140]. 6. Regulation of mammary signals’ emission The emission rate or activity of mammary chemosignals appears to fluctuate intra-individually along the short cycle of nursing and the long cycle of lactation. As mentioned above, the odor mixture carried in lactating rabbits’ abdominal fur releases searching in pups. But pups respond more to females in early, rather than late, lactation [27,62], and in the pre-nursing rather than in the postnursing phase (2 h before vs. after nursing [27]). Thus, the abdominal odor mixture is variable in terms of qualitative and/or quantitative properties. Little is known about the endocrine control of such abdominal odor cues along nursing cycles, and whether it operates in anticipation of nursing or only in response to the actual sucking of pups (or both). Sucking-related tactile stimulation of the nipples is indeed known to releases sharp increases in prolactin and oxytocin [89,152] that control milk production and ejection, and also affect the externalisation of substrates from abdominal sebaceous glands[160]. These endocrine effects of sucking-related somesthesis increase up to 15–20 days post-partum, and then progressively drop in magnitude with incepting weaning [152]. The hormonal control of the tonic emission of the abdominal odor cues has been approached in using ovariectomized females as a model to mimic oestrus, pregnancy and lactation-related levels of pup reactivity. Ovariectomy was first shown to nearly abolish the attractive potency of the abdominal odor of mature females [65]. Administering various combinations of estradiol, progesterone and prolactin to such ovariectomized females led then to the graded increase in pup reactivity to their abdominal odor mixture, maximal searching response level being obtained when the three hormones could act synergistically [51]. To assess whether neonatal reactivity to milk and the pure MP follows fluctuations comparable to those from the whole abdominal odor cues, 2-day-old pups were exposed to the odor of fresh rabbit milk collected from non-related females on postpartum days 2 and 23 (weaning taking place around day 30). The behavioural activity of milk declined between both sampling times. During that same period, the dosage of the MP in the headspace of fresh milk revealed a clear drop in MP concentration [33]. Whether the olfactory attractiveness of the human breast to infants varies as a function of the nursing cycle or of the lactation period remains to be investigated. Nursing-related variations in lactogenic hormones favour indeed milk emission through the main flow or through the miniature lactiferous component of the AG. Likewise, an increased productivity of the sebaceous component of the AG may be effective during late pregnancy and early lactation [18], leading to expectable variations in the composition of areolar secretions. If a communicative function is supported by the AG, one may further expect an increase in their secretory output right after delivery and then before each ensuing nursing bout. Indicative data were obtained on these points: more women tended indeed to evince secretory AG on postpartum days 1–3 than on days 15 or 30 [139]. But more definitive conclusions await further investigation. In the short term of the human nursing cycle, breast attraction to infants may be influenced by local physical changes due to the

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vaso-activity of Haller’s vascular plexus underlying the areolar dermis [97] An acute vasodilatation of this structure may indeed confer a higher surface temperature to the areola as compared to the adjacent skin, hence, accelerating evaporation of odorants present on the areolar surface (especially those oozing from the AG) and presumably optimizing odor release at the time when the infant is offered the breast. This thermal feature of the breast can indeed be anticipatorily triggered by the crying infant [159]. In summary, the best studied case of the rabbit highlights that the production and/or emission of mammary chemosignals are under the tonic control of gonadal steroids and lactogenic hormones. This leads to the externalisation of such chemosignals at the very end of gestation and around birth, when pups’ reactivity to it is at its zenith. Then, at least in the rabbit, chemosignalisation appears to drop: this has been documented for one such signal (the MP) for which release in milk is abolished near weaning. Thus, when lactation comes to an end, rabbit females may handle the weaning conflict by reducing the pup motivation to respond to the MP. Within the period of lactation, the timely control of mammary chemosignalisation is presumably modulated by rhythmic processes (e.g., Ref. [27]) and by infant-related distal and proximal stimuli [159]. 7. Regulation of mammary signals’ perception The decrease in the female’s emission of active odor compounds is echoed in changing reactivity of neonates to them. This has been noted in rabbit pups for the MP. The potency of it to release neonatal responses decreases progressively over the preweaning period, indicating the intervention of poorly understood endogenous and exogenous regulatory factors. The rate of response of domestic pups to the MP goes indeed through several stages, beginning with a first lessening (from >90% to ∼80% respondents) around postnatal days 8–11. A second drop in responsiveness (from ∼80% to ∼40%) occurs between days 11–21. Finally, a major drop is seen after the third postnatal week, when the typical searching–seizing response to the MP vanishes completely by day 28–30 [35,106]. At the same time, the micro-morphology of the response is altered along development [105]. So far this decrease in responsiveness to the MP is explained in correlative terms, relating it to the engagement of competing sensory inputs, improvement in motor skills, increasing metabolic needs, and emergence of capacity to feed autonomously (in terms of oral and digestive processing of vegetal elements). The first fall in MP activity coincides with eye opening (∼days 10–11), which may reorganise the pups’ perceptual balance toward multisensory processing. This is suggested by the increase in directionality of visual and auditory responses (ear lifting) upon MP presentation (from days 10 to 13) [106]. By day 21, pups become mobile, leave the nest box, localise the female visually, initiate suckling, and begin to ingest solid food. Circumstantial observations indicate that, when attempting to suckle, pups appear then to function more on a visual-spatial mode than on a chemosensory mode: when exposed to a supinely laying lactating doe, they target their searching response beneath her (i.e., in direction of the inverted dorsal side) rather than over her (i.e., in direction of the abdominal side bearing the mammary odor cues). Finally, the shift to postlacteal diet correlates with the complete disappearance of the typical cephalic-oral responses to the MP. The rapid bilateral searching actions that allow grasping a fixed nipple are then no more adapted to orally grasp mobile bits of food pellets, grains, or leaves of grass. Interestingly, wild-type pups do not follow the same developmental timing of responsiveness to the MP. Although they are highly reactive to it during the first postnatal week, the first drop also corresponds to the period of eye opening, suggesting again that

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the shift in MP activity depends on the input from non-chemical senses. Otherwise, wild pups’ final drop in responsiveness to the MP is advanced by 10–12 days as compared to domestic pups [35]. In this comparative study, wild pups dispersed around day 20 ([35]; see also [17]), at a time when domestic pups are exposed to peak lactation [129]. The studied samples of wild and domestic females were simultaneously lactating and gestating, hence excluding differential endocrine status and odor emission rate to explain the contrast in pups’ behavioural phenotype. Proximal causes for such a variation may be generally sought in the slower development of domestic breeds selected for prolificacy, increased and extended milk yield, and offspring biomass at birth and weaning. One may also hypothesize that wild pups may be socially canalised to leave the nest earlier to reach feeding autonomy, hence more rapidly curbing down their dependence on the MP. One clue may be in the ecology of the nest itself and related maternal effects. In contrast to their domestic counterparts, wild pups are exposed to a palatable nest lined with grass rather than with a sawdust layer. Intake of such herbal elements already cued in milk or maternal faecal pellets [10], may be a way to accelerate digestive functions from oral processing to caecal decomposition and to nutritional re-uptake through caecotrophy. Further understanding of the developmental context of the phenotypic expression of the milk-food transition in mammals, and related changes and redirections in sensory processing of mammary and food cues, may clearly benefit from research in the rabbit model. The rabbit pup response to mammary/milk signals is also developmentally regulated by processes related to both circadian biology and nutritional/metabolic processes. If one considers pup reactivity to the MP round the clock it changes as a function of milk intake. On day 2, newborn rabbits react automatically, viz. at any time, to the MP, without apparent influences from prior milk intake or from any other emerging circadian factor [103,104]. But by day 5, and more so by day 10, this reliable response to the MP becomes restricted to the pre-nursing hours. The pre-prandial level (5 min before suckling) of MP-released responses decreases from day 2 to day 10 (from >90% to ∼60%responding pups). While no prandial shift in responsiveness to the MP occurs on day 2, an important milk intake-related response decrease (40–50%) is seen on days 5 and 10, with rapid (after 3 h) recuperation of high response rate in the former case and slow recuperation (18 h) in the latter case [104]. These data make clear that nursing behaviour progressively escapes control by the MP with changing metabolic needs and incoming visual information. This shift from automatic to prandially controlled response to the MP is of particular significance in the context of the rare milk resource access evolved by Oryctolagus females. Such response variations over time to milk cues, and specifically to the MP, may warrant that offspring are first tethered to the mammae, and then progressively disinvest them as non-milk foods can be orally and digestively processed. This progressive phenomenon of shifting control from chemosensory to other senses awaits more investigations in mammalian infants. 8. Neonatal strategies to process mammary chemosignals Matching the maternal production of mammary chemosignals, mammalian offspring have co-evolved various mechanisms to process them. Among these mechanisms, some appear to work independently from environmental induction, while others are clearly shaped by exposure effects. Predisposed olfactory processes have been best evidenced in the rabbit newborn, whereas processes involving familiarisation and learning occur in all species examined so far, thus including rabbit and human offspring. It may be suggested from the outset that these mechanisms might be dedicated to process different sets of compounds within the complex mixture composing mammary chemosignals.

8.1. Predisposed strategy: experience-independent responsiveness Organisms respond more intensely to, or learn more easily, certain stimuli than to others. The term ‘predisposed’ applies here when such selective responses emerge without reliance on previous exposure and are resistant to deprivation from the specific stimulus or to its reassignment by other stimuli [14,58]. Such processes designate stimulus-response loops generalised at the species level that are released from birth by stimuli that did not appear to occur in the prior developmental environment. In rabbit pups, odorant stimuli recruit different response levels. When presented for the first time, novel odorants elicit sniffing and often withdrawal reactions. In contrast, odorants that were learned previously can trigger whole body approach, and appetitive and oro-nasal investigation. However, as already mentioned, some biological substrates, such as fresh milk [29,73,138], or some odorants extracted from it, notably the MP [138], release immediate head extension and oral grasping responses despite they were never encountered before. This rapid oral grasping response to fresh milk or the pure MP can be elicited in neonate pups born at or before gestational term (i.e., 1 or 2 days preterm) and deprived thereafter of any contact with a lactating female or her milk. This MP-response loop is general in Oryctolagus newborns from either wild or domestic types, and has thus in principle nothing to do with the local chemosensory ecology (due to genotype- or dietbased metabolic variations) [35,138], or with the psychophysical conditions in which the MP or the reference odorants were presented [30,31]. Finally, the chemical detection of the MP in the amniotic fluid and blood of pregnant females was unsuccessful, indicating that the MP was not present in the prenatal environment, at least according to the current GC detection performances. Thus, the rabbit MP may be considered behaviourally effective from birth without prenatal induction. Another unordinary aspect of the MP response system of rabbit neonates is that it remains highly functional despite long-term exposure deprivation. Separating pups from their mother from birth and bottle-feeding them with a cow milk-based formula devoid of MP for 6 days left their high level responsiveness to the MP unchanged on day 6 [25]. Further, when MP exposuredeprived pups were conditioned to artificial odorants for 6 days, their response rate to the MP remained unaffectedly high and the releasing potency of the newly acquired odorant that recruited the same motor response system (oral grasping) did never surpass that of the MP [25]. Converging processes are suggested to operate in human infants, although any proximate chemosignal could not be pinpointed so far. Newborn infants respond to the odor of human breast (areola) or milk by positive head orientation and appetitive mouthing regardless of the rate of exposure to the breast, i.e., even infants bottle-fed since birth with cow milk- or soy-based formulas express this response [38,84,87,117,127]. Further, when laid prone on a heating mattress and exposed to varying odor conditions, infants exhibit differential approach responses by crawling. When the stimulus is a cotton pad imbued with mother’s breast odor, they move towards it more rapidly than towards a scentless control pad [158]. They even react positively by increased mouthing and sucking movements to conspecific milk odor when born prematurely [11,119], indicating prenatal maturation of related perceptual processes. More recently, we revealed that extremely minute amounts of areolar secretions are also effective in releasing mouthing and in altering respiration in 2-3 day-old infants. This response is specific to AG secretions and effective without prior exposure in the postnatal environment [41]. Much alike the case of neonatal rabbits, the attractive power of conspecific milk odor to human neonates is not easily overcome by newly acquired odorants. This is supported by two series of paired-choice assays comparing the relative attraction to the odors

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of human milk and of artificial odorants associated with breastfeeding. First, when exposed to human milk odor and an artificial odorant (camomile) that was spread on the areolae at each feed since birth, 3-4 day-old breast-feedlings displayed equivalent orientation time to either stimulus [38]. Second, exclusively bottle-fed infants simultaneously presented with the odors of human milk (i.e., from a non-familiar mother) and of their habitual formula turned their head equally towards either stimulus, but additionally they demonstrated more mouthing to the unfamiliar conspecific milk than to the familiar artificial milk [87]. Thus, odor cues carried in human milk appear to be more reinforcing to human newborns than conventional odorants that were repeatedly rewarded by sucking or satiety. However, to date no species-specific unconditionally active compound(s) has been identified in human milk or areolar secretions, which would permit to ascertain that the corresponding perceptual mechanisms have emerged independently from prenatal experience. Thus, in the human case, the predisposed nature (according to the above definition) of the perceptual mechanisms involved in neonatal responses to human milk odor awaits further corroboration. 8.2. Proactive opportunism: responsiveness based on prenatal acquisition The chemosensory systems of fetal and neonatal mammals are unavoidably, passively exposed to odorous by-products of life-sustaining processes produced by the maternal organism. Odorous metabolites can thus be engaged in offspring perceptual activity by virtue of their mere presence in the developmental niches afforded by the mother (amniotic fluid, colostrum/milk, skin surface, and environmental odorants). Their subsequent incentive value depends then on opportunistic associations between chemosensation and arousal states propped up by reinforcements presented by the female. Thus, in parallel with predisposed mechanisms, numerous plastic mechanisms are engaged in the neonatal responsiveness to mammary chemosignals through neuronal selection, synaptic fine-tuning, or learning-based non-associative or associative processes. These plastic processes are shaped opportunistically by the odor conditions prevailing along development, beginning from the fetal period (e.g., Refs. [130,137,145,149,166]). A prenatal impact of odorants on the organism’s olfactory competence supposes that the placental transfer of odorants and their fetal reception is effective, that fetal memory extends into the neonatal period, that amniotic cues are at least in part present in the postnatal environment, and that neonates rely in effect on such prenatally acquired cues. This whole chain of events anticipating selective neonatal behaviour has been documented in both species under consideration. Rabbit pups show indeed positive responses to the odors of freshly collected placentae [29]. Furthermore, when put into a test arena with the opposing odors of fresh placentae and of rabbit milk, newborn pups orient randomly, indicating equivalent sensory treatment of odor stimuli available preand postnatally [29]. Several processes may cause odor similarity between perinatal fluids, the most obvious being the transfer of odorous compounds from the female’s diet into the amnion and colostrum. For example, rabbit pups born to females fed juniper, thyme or cumin seeds subsequently prefer such odorants in spatial or food preference tests [29,145]. Fetal rabbits have thus the capacity to encode in some way an odor encountered in the amniotic fluid, and then, as neonates, remain able to pick it out in the different matrix of milk [29]. Similar processes are at work in human fetuses who, as neonates, remain attracted to volatile compounds met in the amniotic environment [86]. Women who consumed garlic, anise, alcohol or carrot flavour in late pregnancy produce offsprings that manifest posi-

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tive responses towards these odorants, for durations ranging from hours, days to months after birth [48,56,94,136]. But even in the absence of manipulation of the pregnant mother’s dietary flavours, human newborns are able to tell apart the spontaneous odor of their own amniotic fluid from that of an amniotic fluid from another fetus [134,135]. Thus, the olfactory tract and related analytic and memory abilities function well in fetal–neonatal humans [133]. The odor information extracted from the fetal environment is afterwards significant in the context of the mammary niche. Dietary (or other environmental) compounds are indeed physiologically distributed into both amniotic and mammary fluids [55,130], leading to their relative resemblance in volatile composition. Thus, milk-borne odor cues may have gained familiarity before they are directly encountered in colostrum or areolar secretions [130]. 8.3. Reactive opportunism: responsiveness based on postnatal acquisition After birth, newborn organisms intensify their acquisition of any stimulus that is associated with their proximal environment, whether the mother’s body or the nest or sleeping niche. This very active intake of information has been extensively investigated in the rat, in which the developmental course of olfactory abilities, and related cognitive mechanisms and neural underpinnings are best understood [1,13,123,162]. Here, we add data from less-studied rabbit and human neonates, showing that similar forces operate in similar contexts for similar adaptive outcomes. Rabbit pups are remarkably fast in learning odor cues associated with the mother, especially (but not only) when sucking is involved. A series of studies addressed the sensory cues and reinforcing processes that gain control over searching and oral seizing of nipples by pups. From the first day after birth, pups can learn a non-specific odorant associated with nursing [3,34,59,60,69,75]. Learning was established by scenting a female’s ventral fur with varied odorants, letting pups search and suck on her, and then testing them on congruently or differently scented surrogates. It came out that, after only one nursing–odor pairing, pups express the typical sequence of nipple search and attachment on an unfamiliar female or even an anesthetized cat painted with the same odorants [59]. Such learned odorants can remain active for several days after conditioning. But the one-session learning of the odor associated with nipple sucking appears only effective during the first 4 days after birth [75]; after day 5, the nursing-induced odor learning vanishes completely, raising the possibility that the first 4 postnatal days delimit a sensitive period for acquiring odors contingently with suckling. The nursinginduced odor learning appears to follow the same time-course as the odor learning induced by the MP, suggesting that the MP may be instrumental in it. The above results highlight that the context and the act itself of sucking are efficient promoters of odor learning in rabbit newborns. Any arbitrary odor cue which is then sticking on a nipple can be assigned incentive value, and can take signal value for the next suckling episodes [67]. Although sucking may improve the strength of learning in the conditions of the above studies, its realization during the conditioning procedure is unavoidably confounded with exposure to nipple or milk odors, which in themselves can carry strong reinforcers, namely in the form of the MP. Pairing the MP with a neutral odorant (in isolation from the female), without eliciting sucking or arousal, is indeed sufficient to induce the learning of the odorant [34]. Thus, multiple key-agents associated with nipples, or the newborn’s operant responses (i.e., sucking) on them, are to be involved in the early conditioning of mammary odors. Sucking is also influential in prompting learning of odors associated with the breast or milk in human newborns (e.g., Refs. [38,141]), suggesting that natural odors may also be acquired in this way. But again the effect of sucking is not a necessary condition

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as merely exposing newborns to an odorant or delivering them an odor in association with touch-induced arousal suffices to familiarise them to initially irrelevant stimuli [5,151]. Such acquisition of odor cues in human infants seems also subject to modulation by the timing of its occurrence relative to birth [118], but the notion of a sensitive period remains however to be confirmed here. Such facilitated odor learning right after birth had previously been well documented in the rat newborn [79]. In summary, the above results underline that mammalian neonates have evolved multiple ways to process various types of informative odorants, cues which are learned through direct exposure in prior stages of development, and (at least in rabbit pups) signals which are active without any prior exposure. Neonatal behaviour appears thus to be driven by dual olfactory mechanisms supporting either plastic processes calibrated by circumstantial experience in preceding and current environments, and predisposed processes supported by pathways that may be hard-wired to detect species-specific signals. Whether other mammalian species have evolved such perceptual predispositions remains to be ascertained. So far, the human case awaits positive identification of candidate compounds bearing behavioural activity that is unconditional from prior (pre- and postnatal) exposure effects. 9. From behaviour to processing modules: speculations on the neural mediation of the MP The multiple ways of chemosensory processing of mammary messages in newborn mammals–based here on evidence from rabbit and human newborns, but with much more advanced empirical documentation in the former–raise numerous issues about their position and role in the neonatal cognitive architecture, their mapping on neural structures, and how they are altered in the context of rapid maturation and related changes in psychobiological functions (i.e., sensory-motor maturation, increase in metabolic needs, and attainment of feeding self-sufficiency). Regarding the neural systems involved, we can only consider here the rabbit model, in which a simplistic functional division of nasal chemoreception would assume that the MP is processed through the vomeronasal pathway, while common odor cues would be processed through the main olfactory pathway. However, the current, although partial, evidence suggests that both systems mediating predisposed and plastic processes rely on the main olfactory system, and not on the accessory olfactory system. This is supported by the fact that pure MP does not activate the accessory olfactory bulb as assessed by 2-deoxyglucoce (2DG) uptake [128]. Further, the lesion of the vomeronasal pathways does not hinder the expression of typical searching–grasping responses in rabbit pups exposed to the total odor of a lactating female’s abdomen, among which presumably the MP is. However, inactivating the main olfactory mucosa inhibits the pups’ responses to such complex odor cues [63]. Thus, the dissociable predisposed and plastic ways to process odor information in the neonatal rabbit may both be supported by pathways within the main olfactory system (cf. also [8]). This evokes a recently reported case in mice in which predisposed and plastic nasochemosensory processes are also mediated by the main olfactory system. The predisposed fear information carried in trimethyl-thiazoline (faecal fox odor) is indeed forwarded to the brain through a dedicated set of olfactory sensory neurons. When these neurons were genetically ablated, the innate fear response was abolished, but the learning of other odorants remained feasible [76]. Thus, in adult mice, predisposed responses to predator odor and learned aversion responses to food odors are dissociable processes based on distinct pathways within the olfactory bulb. The rabbit case calls for a parallel analysis, adding further elements to the notion of the olfactory bulb built as an assembly of heterogeneous neural subsystems or functional mod-

ules processing compounds of distinct chemical structure (e.g., Refs. [57,71,107,126,156,165]). Separate sets of glomerular clusters reacting more or less exclusively to aliphatic acids, aldehydes, or alcohols, to phenols, or to ketones have indeed been mapped in the adult rat olfactory bulb [108]. Recent data in mice further indicate that some narrowly tuned detectors within the olfactory bulb process urinary chemosignals in parallel with “moderately selective” detectors that process circumstantial, low-intensity odor cues carried in natural mixtures (e.g., Refs. [80,81]). These urine-responsive mitral cells are highly selective (responding to a single component) and localised in two restricted regions of the olfactory bulb. Thus, odor-specific narrowly tuned pathways cohabit with broadly tuned pathways within the mature main olfactory bulb of mammals. The developmental course of such domain-specific glomerular clustering remains poorly understood. However, it is known that glomerular specification increases rapidly in early development. During the first 3 postnatal days, odor-elicited 2DG uptake recruits only a limited set of bulbar zones, whereas by day 15 activated bulbar zones increase and 2DG uptake is massive, close to adult levels [4]. Thus, subsystems within the main olfactory bulb are heterochronous, the earliest being linked with sensory neurons that become functionally mature in advance of others. It may be hypothesised that earlier developing glomeruli may be caught up in detecting and processing stimuli that are of prime importance in the realisation of vital responses in newborn organisms. This extends within a single sensory system the theory that Turkewitz proposed for the whole sensorium of vertebrates. Sensory development starts along a non-random sequence among the different modalities, somesthesic processes being ahead, closely followed by chemosensory, kinaesthetic and auditory processes, and lastly by vision [52]. The consequence of this ordered sensory onset is the sequential “opening” of the brain to sensory experience, which has been viewed as a strategy to reduce the nature, amount and complexity of stimulations available to maturing neural tissue and to regulate competition between emerging sensory systems [155]. The same logic may be applied intra-modally for the development of the main olfactory bulb. A limited set of early functional processing modules may prevent informational overwhelm of the only modality through which altricial neonates can at first efficiently sense their environment. Thus, specialisations in sensory capacities can emerge from such local and temporal differentiation phenomena, and then fade away when more mature states are attained. One such early developing domain-specific cluster of olfactory glomeruli was addressed in the neonatal rat as a specialised structure for detecting mammary cues [153]. This structure, known as the modified glomerular complex (MCG) [53], is anatomically welldefined from late gestation [49] and is strongly activated by suckling cues [53,153]. However, its involvement in sucking-related olfactory competence awaits confirmation [121], which should be facilitated when the odor factor mediating nipple seizing will be identified in the rat. Whether the detection and processing of the rabbit MP is underlain by a dedicated glomerular module (comparable to the rat MCG) or by an “aldehyde module” that is developmentally ahead from others remains to be determined. It may be expected anyway that a bulbar module is at first narrowly tuned to the MP without distracting influence coming from non-pheromonal plastic modules. Such an early functional commitment of processing modules may be a way to ensure that certain stimuli are automatically attended and well connected to a vital behaviour pattern. Both predisposed and plastic olfactory processing modules hypothesised above do however inter-relate during ontogeny. If, as previously outlined, MP-specific predisposed processes in the rabbit pup appear impervious to the influence of odor learning, they are not without effects on odor learning. The predisposed mechanism interacts indeed asymmetrically with plastic olfactory

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mechanisms, the former instructing the latter. Thus, an odor made contingent with the MP incorporates the releasing properties of the MP after only one, very brief (15 s.) pairing episode [34]. Learning a conventional odor in the same conditions without the MP appears comparatively slow (requiring up to 6 associative episodes) [25]. So, although both predisposed and plastic olfactory mechanisms are experimentally dissociable, they are functionally linked. The neural effects of such prompt MP-induced odor learning may be, as abundantly shown in rodent neonates and adults, to accelerate the maturation of wiring and functioning within the olfactory bulb (through selective neuronal apoptosis, neurogenesis, increased dendritic contacts in activated glomerulae that increase in size, increased number of neural cells adjacent to activated glomerulae, increased lateral inhibitory connections and control) and in higher-level structures (cf., e.g., Refs. [16,74,79,122,162]). However, the control of the predisposed mechanism over plastic olfactory mechanisms is time-bound in several ways. First, the MPinduced odor learning functions only during the first 4 postnatal days [32,103,106], demarcating a sensitive period for the initial conversion of odorants incidentally carried in milk or on the mother’s body surface into meaningful cues. Second, as mentioned above, the pheromonal predisposed mechanism shifts from initial automatism in neonatal pups to progressively become controlled by the metabolic needs of growing organisms [104]. Thus, although the MP-related predisposed system mediates maximal responses of pups at any time of the day during the initial period of days 0–4, the response gets more and more tied to internal controls beyond day 4 (i.e., expected nursing rhythm, satiation; [104]). This may suggest that predisposed and plastic mechanisms of olfactory processing are organised in a hierarchical way. At least during an initial period following birth, the predisposed mechanism has either anteriority or priority over plastic mechanisms. Current behavioural evidence suggests that, although the pheromone and prenatallyacquired odors do both trigger appetitive responses at birth (no functional anteriority), the MP has stronger attractive potency than an odorant acquired in utero (functional priority). In summary, in addition to its obvious releasing effect on behaviour, the predisposed pheromonal mechanism is a potent magnifier of olfactory cognition in rabbit pups. During a short but decisive window of early development (postnatal days 1–4), it operates to promote the fast learning of odors that are circumstantially associated with the mother or her milk. This MP-induced learning may be a process through which rabbit newborns can update the odor properties of mammae and milk, which are known to change within a same feed, from feed to feed within a same day, and from day to day (e.g., Ref. [78,88]). Finally, MP-induced odor learning may more generally speed up perceptual tightening by accelerating glomerular refinement [74], and by this way promote the extraction and acquisition of meaningful odors, such as those of the female [115], or nest and littermates [68,146]. This pheromone-enforced learning process evidenced in the rabbit can presently guide us to improve our understanding of how mammalian neonates decode and encode chemosensory information from exceedingly complex and changing stimuli [36]. It should also stimulate the determination of the precise nature and development of the neural structures involved in the olfactory processing of the MP and of conventional odor cues. Dedicated neural pathways to process mammary chemosignals in the rabbit newborn, if any, would then prove informative about similar possibilities in the newborns of other species, including our own. 10. Conclusions Mammals have undergone selective pressure enforcing their neonates to attend biologically meaningful stimuli that direct them to the maternal body and the source of milk with minimal distrac-

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tion from the “blooming–buzzing confusion” of incoming stimuli. One general strategy has been to limit the range of effective sensory systems [155], so that only parts of the multisensory perceptual apparel are involved in controlling adaptive behaviour (viz., mainly chemosensation and somesthesis in species with altricial newborns such as those of rabbits; or somesthesis, chemoreception and audition in precocial newborns, such as those of humans). Another strategy has been to specify within a same functioning sensory system predisposed subsystems dedicated to process species-specific stimuli that are immediately salient for newborns. The rabbit MP is a case of such a stimulus appearing to be processed by a predisposed olfactory subsystem that elicits an automatic, stereotyped sequence of responses. Finally, the most ubiquitous strategy has been to rapidly impinge salience to incidental stimuli through experience-sensitive processes, resulting in neonatal detection and response capabilities that are closely tailored to the conditions of the local environment. Such experience-dependent processes are in fact trophic to the concerned sensory system in the earliest phases of development so that they shape its functional properties i) before actual involvement in adaptive responses (i.e., prenatal induction of receptor expression, familiarization and learning), as well as ii) during/after the realisation of such responses (i.e., continued postnatal shaping of olfactory structures and functioning). As seen above, these different cognitive strategies may interact during sensitive periods, as in the rabbit newborn in which a predisposed mechanism commands plastic mechanisms to engage associative processes only during the first 4 postnatal days. So that during this sensitive window, neonatal rabbits can do nothing but learn the odorants that stick on the mother or the wider environment (nest or littermates). Thus, the mechanisms described above are certainly not mutually exclusive: they work in a redundant, cooperative manner leading to the over-determined learning of stimuli that are contingent with the females, their mammae and milk. These multiple ways to process mammary chemosignals have been most investigated in rabbit pups, following the wave of extensive research on related issues in rodent neonates (reviewed in [12,13,15,150,154]). Oryctolagus newborns appear indeed to be endowed with the whole range of perceptual/cognitive processes that optimise the rapid engagement of olfaction in the control of nursing behaviour: they respond right after birth to an unconditional pheromonal stimulus (the mammary pheromone), to odorants acquired as cues prenatally, and they can very quickly learn additional odor cues postnatally. The same range of chemocognitive processes are functional in rat newborns (although no mammary or milk-related pheromone has been definitely identified so far in rats) which develop in radically different conditions of mother–offspring relations [12]. While rat females stay with their litter for more than 70% during the first postpartum days [124], rabbit females invest less than 0,1% in direct contact with their newborn pups [167]. Although in either species newborns are apparently not exposed to the same level of hurry to find a nipple in terms of mother’s accessibility, they face however the same imperative need to take in colostral energy, immuno-protection, and promnesic boost immediately after birth. It is probably the immediacy of this vital need that represents the ultimate cause that led rabbit and rat females to convergently exploit all perceptual, reactive and cognitive resources in their altricial newborns, namely i) in favouring the integrated use of distal (olfaction) and proximal (perioral somesthesis) sensing of the mammary surface, and ii) in taking advantage of the multiple cognitive solutions that could be mediated by the most functional distance (teloceptive) sense, olfaction. Whether the same range of cognitive solutions to the vital challenge of initial colostrum/milk intake is effective in neonates from other mammalian species, namely species producing precocial neonates and specifically humans, remain to be empirically

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established. Again, despite huge inter-specific variation in the structure and dynamics of the human female–neonate bond, the similarity of selective forces exerted on both partners in the dyad around birth may predict convergent solutions. The above-surveyed data from human neonates suggest indeed that olfaction-related processes may also operate in the nursing context. But the existence of species-specific mammary chemosignals and the predisposed nature of corresponding processing modules and response systems could not be ascertained so far. In sum, mammals offer a fertile ground for comparative investigations on the chemosensory controls of female–offspring communication. Such an endeavour should come out to specify the range of perceptual, cognitive, and behavioural solutions coevolved by mammalian females and neonates, and to assess how far they were evolutionarily elaborated in response to species-specific constraints regarding the critical procurement and intake of milk. Acknowledgments The preparation of this review and the experimental work from our research group reported therein was mainly supported by CNRS and Etablissement National d’Enseignement Supérieur Agricole, Dijon, France. It also benefited from grants from the French Ministry of Research and Technology (ACI ‘Neurosciences Intégratives et Computationnelles’); Regional Councils of Poitou-Charentes and of Burgundy; Agence Nationale pour la Recherche; Nestec, Lausanne, Switzerland; and the European Community (COST 848). The contributions of Guy Perrier, Jean-Pierre Drouet, Monique Jouanno, Dominique Langlois, Etienne Sémon, Jean-Luc Le Quéré, Robyn Hudson, Pierre Coudert, Laurence Fortun-Lamothe, Gilles Sicard, Ingrid Jakob, Liliane Astic, Diane Saucier, Brigitte and André Quénnedey, and Heiko G Rödel are acknowledged for the rabbit work; for the human work, we thank especially Robert Soussignan, Paul Sagot, Tao Jiang, Luc Marlier and Elisabeth Hertling. References [1] Alberts JR. Early learning and ontogenetic adaptation. In: Krasnegor NA, Blass EM, Hofer MA, Smotherman WP, editors. Perinatal development, a psychobiological perspective. Orlando, FA: Academic Press; 1987. p. 12–38. [2] Albone ES. Mammalian semiochemistry. Chichester, UK: Wiley; 1984. [3] Allingham K, Brennan PA, Distel H, Hudson R. Expression of c-Fos in the main olfactory bulb of neonatal rabbits in response to garlic as a novel and conditioned odor. Behav Brain Res 1999;104:157–67. [4] Astic L, Saucier D. Ontogenesis of the functional activity of rat olfactory bulb: autoradiographic study with the 2-deoxyglucose method. Dev Brain Res 1982;2:1243–56. [5] Balogh J, Porter RH. Olfactory preferences resulting from mere exposure in human neonates. Infant Behav Dev 1986;9:395–401. [6] Bautista A, Mendoza-Degante M, Coureaud G, Martina-Gomez M, Hudson R. Scramble competition in newborn domestic rabbits for an unusually limited milk supply. Anim Behav 2005;70:1011–21. [7] Bautista A, Garcia-Torres E, Martinez-Gomez M, Hudson R. Do newborn domestic rabbits Oryctolagus cuniculus compete for thermally advantageous positions in the litter huddle? Behav Ecol Sociobiol 2008;62:331–9. [8] Baxi KN, Dories KM, Eisthen HL. Is the vomeronasal system really specialised for detecting pheromones? Trends Neurosci 2006;29:1–7. [9] Beauchamp GK, Doty RL, Moulton DG, Mugford RA. The pheromone concept in mammals: a critique. In: Doty RL, editor. Mammalian olfaction, reproductive processes, and behavior. New York: Academic Press; 1976. p. 143–60. [10] Bilko A, Altbäcker V, Hudson R. Transmission of food preference in the rabbit: the means of information transfer. Physiol Behav 1994;56:907–12. [11] Bingham PM, Abassi S, Sivieri E. A pilot study of milk odor effect on nonnutritive sucking by premature infants. Arch Pediatr Adolesc Med 2003;157:72–5. [12] Blass EM. Suckling: determinants, changes, mechanisms, and lasting impressions. Dev Psychol 1990;26:520–33. [13] Blass EM, Teicher MH. Suckling. Science 1980;210:15–22. [14] Bolhuis JJ. Development of perceptual mechanisms in birds: predispositions and imprinting. In: Moss CF, Shettleworth SJ, editors. Neuroethological studies of cognitive and perceptual processes. Boulder (Colorado): Westview Press; 1996. p. 158–84. [15] Brake SC, Shair H, Hofer MA. Exploiting the nursing niche: The infant’s sucking and feeding in the context of the mother-infant interaction. In: Blass EM, editor. Developmental psychobiology and behavioral ecology, Handbook of behavioral neurobiology, Vol. 9. New York: Plenum; 1986. p. 347–88.

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