A pheromone to behave, a pheromone to learn: the rabbit mammary pheromone

Birth is part of a continuum and is a major developmental change. Newborns need to adapt rapidly to the environment in terms of physiology and behaviour, and ability to locate the maternal source of milk is vital. Mechanisms have evolved resulting in the emission of olfactory cues by the mother and the processing of these cues by the young. Here, we focus on some sensory, cognitive and behavioural strategies developed by the European rabbit (Oryctolagus cuniculus) that optimize the early development of offspring. In this species, chemosensory communication between the mother and young plays a critical role in eliciting adaptive neonatal responses. In particular, lactating females release a molecule, the mammary pheromone, which has several functional impacts. It triggers orocephalic responses involved in the quick localization of nipples and sucking. Moreover, this unconditioned signal promotes rapid appetitive learning of novel odorants, acting as a potent organizer of neonatal cognition. The mammary-pheromone-induced odour memory requires consolidation/reconsolidation processes to be maintained in the long term. Finally, as this mode of conditioning also promotes learning of mixtures of odorants, it supports investigations related to the capacity of neonatal olfaction to extract biological value from the complex environment.     

The reactivity of neonatal rabbits to the mammary pheromone as a probe for viability

Newborn rabbits depend on a daily nursing interaction with the mother to gain milk and to survive. During this interaction, they localise and seize the nipples displaying a typical behaviour triggered by maternal odour cues. The mammary pheromone constitutes such a signal in domestic rabbits: it elicits sucking-related movements in more than 90% of the pups. However, some newborns remain unresponsive to the presentation of the pheromone, even pups apparently healthy and highly motivated to suck. The main goal of the present study was therefore to explore the link between the unresponsiveness of rabbit pups to the mammary pheromone and their growth and survival in breeding conditions. To that end, 293 newborns from 30 litters were tested for their head searching-oral grasping responses to the mammary pheromone on days 1 and 3, and their milk intake and mortality were followed up from days 1 to 21. It was hypothesised that unresponsive newborns would have subsequent difficulties in finding nipples, sucking and surviving. Early weight and success in milk intake were further considered as mediating factors in growth and viability. The results showed that pups that were unresponsive to the mammary pheromone on day 1 were less successful in gaining milk and had a higher rate of mortality than the responsive pups. However, this impact was modulated by the weight of pups: it appeared only in the lightest newborns. Moreover, this impact vanished on day 3. On the other hand, the pup weight and sucking success on days 1 to 3 strongly influenced viability and growth during the period extending from days 1 to 21. Taken together, the results show that the day-1 responsiveness of rabbit pups to the mammary pheromone can be considered as an indicator of individual viability in pups having a small weight (<48 g on day 1). The predictive validity of the pups’ pheromonal reactivity seems however time-limited as it works only during the first, but crucial, postnatal days.     

Got milk? A pheromonal message for newborn rabbits

A substance in rabbit milk, 2-methylbut-2-enal (2MB2), has been identified as a pheromone that triggers stereotypical searching behavior from rabbit pups. Pups respond to the odor of 2MB2 solutions in concentration-dependent manner, but fail to respond to 20 other volatile components in rabbit milk and 20 additional odorants. The effectiveness of 2MB2 generalizes across strains and breeds of rabbits, but is ineffective in closely related species. Finally, pup responsiveness to 2MB2 is innate and does not require learning. This study, for the first time, identifies a mammary pheromone that provides sufficient sensory cue for nipple attachment by newborns. In addition to contributing to our understanding of pheromonal communication, it provides an advantageous model system for neurobiologists.     

Cross-generational impact of a male murine pheromone 2-sec-butyl-4,5-dihydrothiazole in female mice

The current understanding of the activity of mammalian pheromones is that endocrine and behavioural effects are limited to the exposed individuals. Here, we demonstrate that the nasal exposure of female mice to a male murine pheromone stimulates expansion of mammary glands, leading to prolonged nursing of pups. Subsequent behavioural testing of the pups from pheromone-exposed dams exhibited enhanced learning. Sialic acid components in the milk are known to be involved in brain development. We hypothesized that the offspring might have received more of this key nutrient that promotes brain development. The mRNA for polysialyltransferase, which produces polysialylated neural cell adhesion molecules related to brain development, was increased in the brain of offspring of pheromone-exposed dams at post-natal day 10, while it was not different at embryonic stages, indicating possible differential brain development during early post-natal life.     

Characterization of an alarm pheromone secreted by amphibian tadpoles that induces behavioral inhibition and suppression of the neuroendocrine stress axis

Many species assess predation risk through chemical cues, but the tissue source, chemical nature, and mechanisms of production or action of these cues are often unknown. Amphibian tadpoles show rapid and sustained behavioral inhibition when exposed to chemical cues of predation. Here we show that an alarm pheromone is produced by ranid tadpole skin cells, is released into the medium via an active secretory process upon predator attack, and signals predator presence to conspecifics. The pheromone is composed of two components with distinct biophysical properties that must be combined to elicit the behavioral response. In addition to the behavioral response, exposure to the alarm pheromone caused rapid and strong suppression of the hypothalamo-pituitary-adrenal (HPA) axis, as evidenced by a time and dose-dependent decrease in whole body corticosterone content. Reversing the decline in endogenous corticosterone caused by exposure to the alarm pheromone through addition of corticosterone to the aquarium water (50 nM) partially blocked the anti-predator behavior, suggesting that the suppression of the HPA axis promotes the expression and maintenance of a behaviorally quiescent state. To our knowledge this is the first evidence for aquatic vertebrate prey actively secreting an alarm pheromone in response to predator attack. We also provide a neuroendocrine mechanism by which the behavioral inhibition caused by exposure to the alarm pheromone is maintained until the threat subsides.     

Orientation of male gypsy moths,Lymantria Dispar (L.), to pheromone sources: The role of olfactory and visual cues

The role of olfaction and vision in the close-ranging flying and walking orientation of male gypsy moths, Lymantria dispar(L.), to females was studied in the forest and in the laboratory. In the forest, feral males found an isolated pheromone source as readily as one supplemented with female visual cues; dead, acetonerinsed females deployed without pheromone received virtually no visitations. In flight tunnel choice experiments using cylinders as surrogate trees and pheromone in different spatial configurations, visual attributes of the female did not influence either the males' choice of landing site or the efficiency with which they located the female. Rather, the presence of pheromone on the cylinder was necessary to elicit orientation as well as landing and walking on the cylinder. When a female visual model was placed in various positions around a pheromone source, walking males oriented primarily to the chemical stimulus. There were, however, indications that males would alter their walking paths in response to female visual cues over short distances (<5 cm), but only if they continued to receive pheromone stimulation. When visual and chemical cues were abruptly uncoupled by altering the trajectory of the pheromone plume, most males responded to the loss of the odor cue rather than to visual cues from the female. Temporal pheromone stimulation patterns affected male walking orientation. When stimulated by pheromone, males oriented toward the source; loss of the odor cue prompted an arearestricted local search characterized by primarily vertical and oblique movements with frequent reversals in direction. Presumably these maneuvers enhance the likelihood of recontacting the plume or serendipitously encountering the female. The apparent lack of visual response to the female is discussed in light of morphological and behavioral evidence suggesting that gypsy moths were formerly nocturnal.     

Mixture interactions in moth olfactory physiology: examining the effects of odorant mixture, concentration, distal stimulation, and antennal nerve transection on sensillar responses

The insect olfactory system is challenged to decipher valid signals from among an assortment of chemical cues present in the airborne environment. In the moth, Heliothis virescens, males rely upon detection and discrimination of a unique blend of components in the female sex pheromone to locate mates. The effect of variable odor mixtures was used to examine physiological responses from neurons within sensilla on the moth antenna sensitive to female sex pheromone components. Increasing concentrations of heliothine sex pheromone components applied in concert with the cognate stimulus for each neuronal type resulted in mixture suppression of activity, except for one odorant combination where mixture enhancement was apparent. Olfactory receptor neuron (ORN) responses were compared between moths with intact and transected antennal nerves to determine whether specific instances of suppression might be influenced by central mechanisms. Type A sensilla showed little variation in response between transected and intact preparations; however, recordings from type B sensilla with transected antennal nerves exhibited reduced mixture suppression. Testing by parallel stimulation of distal antennal segments while recording and stimulating proximal segments dismissed the possibility of interneuronal or ephaptic effects upon sensillar responses. The results indicate that increasing concentrations of "noncognate" odorants in an odor mixture or antennal nerve transection can produce variation in the intensity and temporal dynamics of physiological recordings from H. virescens ORNs.     

Sensory Aspects of Trail-Following Behaviors in the Asian Longhorned Beetle, <Emphasis Type="Italic">Anoplophora glabripennis</Emphasis>

Anoplophora glabripennis has a complex suite of mate-finding behaviors, the functions of which are not entirely understood. These behaviors are elicited by a number of factors, including visual and chemical cues. Chemical cues include a male-produced volatile semiochemical acting as a long-range sex pheromone, a female-produced cuticular hydrocarbon blend serving as a sex-identification contact pheromone, and a recently identified female-produced trail sex pheromone that is followed by mate-seeking males. However, the sensory appendages and sensilla on these appendages used to detect the trail sex pheromone are unknown. We evaluated the ability of virgin male A. glabripennis to follow a sex pheromone trail after removal of the terminal four antennal segments and/or the maxillary and labial palps using a two-choice behavioral bioassay. We also tested the ability of males to follow the trail sex pheromone using volatile pheromone cues only, without physical contact with the pheromone. Results indicate that the palps are primarily responsible for sensing the pheromone, with males lacking palps unable to respond behaviorally to the trail sex pheromone. Under the conditions of this study, males could not follow the sex pheromone trail without direct contact, suggesting that olfaction may not be involved in detection of this pheromone. However, we did not determine to what degree the trail pheromone chemicals can volatilize under our experimental conditions. This work is important in elucidating the behaviors and sensory structures involved in mate-finding by this species on host trees, and these studies may help determine whether the trail sex pheromone has applications for monitoring and management.     

Honeybees Learn Odour Mixtures via a Selection of Key Odorants

Background

The honeybee has to detect, process and learn numerous complex odours from her natural environment on a daily basis. Most of these odours are floral scents, which are mixtures of dozens of different odorants. To date, it is still unclear how the bee brain unravels the complex information contained in scent mixtures.

Methodology/Principal Findings

This study investigates learning of complex odour mixtures in honeybees using a simple olfactory conditioning procedure, the Proboscis-Extension-Reflex (PER) paradigm. Restrained honeybees were trained to three scent mixtures composed of 14 floral odorants each, and then tested with the individual odorants of each mixture. Bees did not respond to all odorants of a mixture equally: They responded well to a selection of key odorants, which were unique for each of the three scent mixtures. Bees showed less or very little response to the other odorants of the mixtures. The bees'' response to mixtures composed of only the key odorants was as good as to the original mixtures of 14 odorants. A mixture composed of the other, non-key-odorants elicited a significantly lower response. Neither an odorant''s volatility or molecular structure, nor learning efficiencies for individual odorants affected whether an odorant became a key odorant for a particular mixture. Odorant concentration had a positive effect, with odorants at high concentration likely to become key odorants.

Conclusions/Significance

Our study suggests that the brain processes complex scent mixtures by predominantly learning information from selected key odorants. Our observations on key odorant learning lend significant support to previous work on olfactory learning and mixture processing in honeybees.     

Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein

Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSH(D118A), produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSH(D118A) reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants.     

Proportion of odorants impacts the configural versus elemental perception of a binary blending mixture in newborn rabbits

Processing of odor mixtures by neonates is weakly understood. Previous studies showed that a binary mixture of ethyl isobutyrate/ethyl maltol (odorants A/B) blends in newborn rabbits at the 30/70 ratio: Pups would perceive a configural odor in addition to the components' odors. Here, we investigated whether the emergence of this additional odor in AB is determined by specific ratio(s) of A and B. To that goal, we tested whether pups discriminated between AB mixtures with lower (A(-)B, 8/92 ratio) or higher (A(+)B, 68/32) proportion of A. In Experiment 1, pups conditioned to A (or B) responded to A(-)B and A(+)B but not to AB. In Experiment 2, pups responded to A(-)B after learning of A(-) (and to A(+)B after learning of A(+)) but not to AB. In Experiment 3, after conditioning to A(-)B pups responded to A(-) and B (and to A(+) and B after learning of A(+)B) but not or less to AB. In Experiment 4, pups responded to A(-)B and A(+)B after conditioning to AB. These results confirm the configural perception of certain odor mixtures by young organisms and reveal that the proportion of components is a key factor influencing their coding, recognition, and discrimination of complex stimuli.     

Distinct haptic cues do not reduce interference when learning to reach in multiple force fields

Background

Previous studies of learning to adapt reaching movements in the presence of novel forces show that learning multiple force fields is prone to interference. Recently it has been suggested that force field learning may reflect learning to manipulate a novel object. Within this theoretical framework, interference in force field learning may be the result of static tactile or haptic cues associated with grasp, which fail to indicate changing dynamic conditions. The idea that different haptic cues (e.g. those associated with different grasped objects) signal motor requirements and promote the learning and retention of multiple motor skills has previously been unexplored in the context of force field learning.

Methodology/Principle Findings

The present study tested the possibility that interference can be reduced when two different force fields are associated with differently shaped objects grasped in the hand. Human subjects were instructed to guide a cursor to targets while grasping a robotic manipulandum, which applied two opposing velocity-dependent curl fields to the hand. For one group of subjects the manipulandum was fitted with two different handles, one for each force field. No attenuation in interference was observed in these subjects relative to controls who used the same handle for both force fields.

Conclusions/Significance

These results suggest that in the context of the present learning paradigm, haptic cues on their own are not sufficient to reduce interference and promote learning multiple force fields.     

Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate

Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male's strategy to suppress reproduction by competing males.     

Olfactory perception in honeybees: Concatenated and mixed odorant stimuli,concentration, and exposure effects

Summary Here we present results obtained from 7 different series of experiments, all employing odor conditioning of proboscis extension in worker honeybees and each designed to address a particular question involving olfactory perception. The questions relate to: temporal complexity of odor cues; effects of concentration, suppression, and/or potentiation in mixture perception; acquisition and extinction rates, as well as levels of generalization associated with aliphatic compounds that have the same functional groups or same alkyl radical length; and the effects of continuous exposure to odorants in the first several days of adult life on various learning and discrimination tasks involving olfactory perception. From the data obtained in these experiments we were able to conclude the following: First, worker honeybees have a limited ability to perceive complex temporal odor-quality patterns in olfactory stimuli — they learn to associate the quality of only the last part of the stimulus with a sucrose reward. Second, we confirm that citral is qualitatively different in several perceptual contexts involving odor learning and conditioning and our results help elucidate the nature of these differences as they relate to learning, discrimination, mixture perception, and continuous exposure to particular odorants. Third, we appear to have uncovered some important perceptual differences between functional groups attached to the first as opposed to the second carbon atom of alkyl radicals. Finally, we failed to uncover any significant effects relating to continuous exposure to odorants during the first several days of a worker's adult life, despite evidence that considerable sensory development takes place during this period. Thus ontogenetic changes to the peripheral system due to environmental effects appear to leave basic perceptual systems unaltered.     

Using strategic movement to calibrate a neural compass: a spiking network for tracking head direction in rats and robots

The head direction (HD) system in mammals contains neurons that fire to represent the direction the animal is facing in its environment. The ability of these cells to reliably track head direction even after the removal of external sensory cues implies that the HD system is calibrated to function effectively using just internal (proprioceptive and vestibular) inputs. Rat pups and other infant mammals display stereotypical warm-up movements prior to locomotion in novel environments, and similar warm-up movements are seen in adult mammals with certain brain lesion-induced motor impairments. In this study we propose that synaptic learning mechanisms, in conjunction with appropriate movement strategies based on warm-up movements, can calibrate the HD system so that it functions effectively even in darkness. To examine the link between physical embodiment and neural control, and to determine that the system is robust to real-world phenomena, we implemented the synaptic mechanisms in a spiking neural network and tested it on a mobile robot platform. Results show that the combination of the synaptic learning mechanisms and warm-up movements are able to reliably calibrate the HD system so that it accurately tracks real-world head direction, and that calibration breaks down in systematic ways if certain movements are omitted. This work confirms that targeted, embodied behaviour can be used to calibrate neural systems, demonstrates that 'grounding' of modelled biological processes in the real world can reveal underlying functional principles (supporting the importance of robotics to biology), and proposes a functional role for stereotypical behaviours seen in infant mammals and those animals with certain motor deficits. We conjecture that these calibration principles may extend to the calibration of other neural systems involved in motion tracking and the representation of space, such as grid cells in entorhinal cortex.     

The effect of salinity on the chemotaxis of glass eels, Anguilla anguilla, to organic earthy and green odorants

Synopsis The behaviour of upstream migrating glass eels to salt and brackish water solutions of 8 pure organic earthy and green odorants was investigated. In 35 salt water, IPMCET and D-MF are ineffective, while MMP, IBMP, MT, TMCE, ETMCE and L-MF are strongly repellent. Dissolved in brackish water, MMP and ETMCE become attractive. The strong attraction which glass eels show to earthy and green odorants emerges as the level of salinity is reduced, suggesting that these chemicals could be orienting cues in the last phase of glass eel migration.     

Newborn rabbit responsiveness to the mammary pheromone is concentration-dependent

The effect of the intensity of odour signals has rarely been investigated in the regulation of odour-guided behaviour in young mammals. This series of experiments used the mammary pheromone (MP) of the female rabbit to assess the influence of stimulus concentration on neonatal pup responsiveness. The MP is a single compound isolated from rabbit milk that releases in pups the typical head searching and oral seizing behaviour. The pups (n = 621) were exposed to graded concentrations of the MP in bioassays varying in stimulus delivery conditions. Experiment 1 demonstrated that in aqueous dilutions the MP efficiently elicits behavioural responses only within a limited range of concentrations (from 2.5 x 10(-9) to 2.5 x 10(-5) g/ml). Experiment 2 yielded the same outcome with highly purified MP delivered in dynamic conditions with a gas chromatograph. Finally, Experiment 3 used deodorized milk as the solvent of the MP; despite this change in the physico-chemical context of stimulation, similar results were reached.