Centrifugal projections to the main olfactory bulb revealed by transsynaptic retrograde tracing in mice.

A wide range of evidence indicates that olfactory perception is strongly involved in food intake. However, the polysynaptic circuitry linking the brain areas involved in feeding behavior to the olfactory regions is not well known. The aim of this article was to examine such circuits. Thus, we described, using hodological tools such as transsynaptic viruses (PRV152) transported in a retrograde manner, the long-distance indirect projections (two to three synapses) onto the main olfactory bulb (MOB). The ß-subunit of the cholera toxin which is a monosynaptic retrograde tracer was used as a control to be able to differentiate between direct and indirect projections. Our tracing experiments showed that the arcuate nucleus of the hypothalamus, as a major site for regulation of food intake, sends only very indirect projections onto the MOB. Indirect projections to MOB also originate from the solitary nucleus which is involved in energy homeostasis. Other indirect projections have been evidenced in areas of the reward circuit such as VTA and accumbens nucleus. In contrast, direct projections to the MOB arise from melanin-concentrating hormone and orexin neurons in the lateral hypothalamus. Functional significances of these projections are discussed in relation to the role of food odors in feeding and reward-related behavior.

Brain anatomy of the 4-day-old European rabbit.

The European rabbit (Oryctolagus cuniculus) is a widely used model in fundamental, medical and veterinary neurosciences. Besides investigations in adults, rabbit pups are relevant to study perinatal neurodevelopment and early behaviour. To date, the rabbit is also the only species in which a pheromone - the mammary pheromone (MP) - emitted by lactating females and active on neonatal adaptation has been described. The MP is crucial since it contributes directly to nipple localisation and oral seizing in neonates, i.e. to their sucking success. It may also be one of the non-photic cues arising from the mother, which stimulates synchronisation of the circadian system during pre-visual developmental stages. Finally, the MP promotes neonatal odour associative and appetitive conditioning in a remarkably rapid and efficient way. For these different reasons, the rabbit offers a currently unique opportunity to determine pheromonal-induced brain processing supporting adaptation early in life. Therefore, it is of interest to create a reference work of the newborn rabbit pup brain, which may constitute a tool for future multi-disciplinary and multi-approach research in this model, and allow comparisons related to the neuroethological basis of social and feeding behaviour among newborns of various species. Here, in line with existing experimental studies, and based on original observations, we propose a functional anatomical description of brain sections in 4-day-old rabbits with a particular focus on seven brain regions which appear important for neonatal perception of sensory signals emitted by the mother, circadian adaptation to the short and single daily nursing of the mother in the nest, and expression of specific motor actions involved in nipple localisation and milk intake. These brain regions involve olfactory circuits, limbic-related areas important in reward, motivation, learning and memory formation, homeostatic areas engaged in food anticipation, and regions implicated in circadian rhythm and arousal, as well as in motricity.

Morphology and evolution of the oral shield in marsupial neonates including the newborn monito del monte (Dromiciops gliroides, Marsupialia Microbiotheria) pouch young.

Newborn marsupials can be arranged into three grades of developmental complexity based on their external form, as well as based on their organ systems and their cytology. The dasyurids are considered the least developed marsupials at birth, while didelphids and peramelids are intermediate, and macropods are the most developed. Currently there is still little information on caenolestid and microbiotherid development at birth. Developmental stages can be graded as G1, G2 and G3, with G1 being the least developed at birth, and G3 the most developed. Marsupials are also characterized by having an extremely developed craniofacial region at birth compared with placentals. However, the facial region is also observed to vary in development between different marsupial groups at birth. The oral shield is a morphological structure observed in the oral region of the head during late embryological development, which will diminish shortly after birth. Morphological variation of the oral shield is observed and can be arranged by developmental complexity from greatly developed, reduced to vestigial. In its most developed state, the lips are fused, forming together with the rhinarium, a flattened ring around the buccal opening. In this study, we examine the external oral shield morphology in different species of newborn marsupials (dasyurids, peramelids, macropods and didelphids), including the newborn monito del monte young (Dromiciops gliroides - the sole survivor of the order Microbiotheria). The adaptive value of the oral shield structure is reviewed, and we discuss if this structure may be influenced by developmental stage of newborn, pouch cover, species relatedness, or other reproductive features. We observe that the oral shield structure is present in most species of Marsupialia and appears to be exclusively present in this infraclass. It has never been described in Monotremata or Eutherians. It is present in unrelated taxa (e.g. didelphids, dasyurids and microbiotherids). We observe that a well-developed oral shield may be related to ultra altricial development at birth, large litter size (more than two), and is present in most species that lack a pouch in reproductive adult females or have a less prominent or less developed pouch with some exceptions. We try to explore the evolution of the oral shield structure using existing databases and our own observations to reconstruct likely ancestral character states that can then be used to estimate the evolutionary origin of this structure and if it was present in early mammals. We find that a simple to develop oral shield structure (type 2-3) may have been present in marsupial ancestors as well as in early therians, even though this structure is not present in the extant monotremes. This in turn may suggest that early marsupials may have had a very simple pouch or lacked a pouch as seen in some living marsupials, such as some dasyurids, didelphids and caenolestids. The study's results also suggest that different morphological stages of the oral shield and hindlimb development may be influenced by species size and reproductive strategy, and possibly by yet unknown species-specific adaptations.

Spontaneous brain processing of the mammary pheromone in rabbit neonates prior to milk intake.

Chemical signals play a critical role in interindividual communication, including mother-young relationships. Detecting odor cues released by the mammary area is vital to the newborn's survival. European rabbit females secret a mammary pheromone (MP) in their milk, which releases sucking-related orocephalic movements in newborns. Pups spontaneously display these typical movements at birth, independently of any perinatal learning. Our previous Fos mapping study (Charra et al., 2012) performed in 4-day-old rabbits showed that the MP activated a network of brain regions involved in osmoregulation, odor processing and arousal in comparison with a control odor. However, at this age, the predisposed appetitive value of the MP might be reinforced by previous milk intake. Here, the brain activation induced by the MP was examined by using Fos immunocytochemistry and compared to a neutral control odor in just born pups (day 0) that did not experienced milk intake. Compared to the control odor, the MP induced an increased Fos expression in the posterior piriform cortex. In the lateral hypothalamus, Fos immunostaining was combined with orexin detection since this peptide is involved in arousal/food-seeking behavior. The number of double-labeled cells was not different between MP and control odor stimulations but the total number of Fos stained cells was increased after MP exposure. Our results indicate that the MP does not activate the same regions in 0- vs. 4-day-old pups. This difference between the two ages may reflect a changing biological value of the MP in addition to its constant predisposed releasing value.

Brain processing of a configural vs elemental odor mixture in the newborn rabbit.

Organisms are surrounded throughout life by chemically complex odors. How individuals process an odorant within a mixture or a mixture as a whole is a key question in neuroethology and chemical senses. This question is addressed here by using newborn rabbits, which can be rapidly conditioned to a new stimulus by single association with the mammary pheromone. After conditioning to ethyl maltol (odorant B), pups behaviorally respond to B and an A'B' mixture (68/32 ratio) but not to ethyl isobutyrate (odorant A) or an AB mixture (30/70 ratio). This suggests elemental and configural perception of A'B' and AB, respectively. We then explored the neural substrates underlying the processing of these mixtures with the hypothesis that processing varies according to perception. Pups were pseudoconditioned or conditioned to B on postnatal day 3 before exposure to B, A'B' or AB on day 4. Fos expression was not similar between groups (mainly in the olfactory bulb and posterior piriform cortex) suggesting a differential processing of the stimuli that might reflect either stimulus complexity or conditioning effect. Thus, the ratio of components in A'B' vs AB leads to differential activation of the olfactory system which may contribute to elemental and configural percepts of these mixtures. In addition, together with recent behavioral data, this highlights that configural perception occurs even in relatively immature animals, emphasizing the value of the newborn rabbit for exploration of odor mixture processing from molecules to brain and behavior.

Goα expression in the vomeronasal organ and olfactory bulb of the tammar wallaby.

The vomeronasal organ (VNO) detects pheromones via 2 large families of receptors: vomeronasal receptor 1, associated with the protein Giα2, and vomeronasal receptor 2, associated with Goα. We investigated the distribution of Goα in the developing and adult VNO and adult olfactory bulb of a marsupial, the tammar wallaby. Some cells expressed Goα as early as day 5 postpartum, but by day 30, Goα expressing cells were distributed throughout the receptor epithelium of the VNO. In the adult tammar, Goα appeared to be expressed in sensory neurons whose nuclei were mostly basally located in the vomeronasal receptor epithelium. Goα expressing vomeronasal receptor cells led to all areas of the accessory olfactory bulb (AOB). The lack of regionally restricted projection of the vomeronasal receptor cell type 2 in the tammar was similar to the uniform type, with the crucial difference that the uniform type only shows expression of Giα2 and no expression of Goα. The observed Goα staining pattern suggests that the tammar may have a third accessory olfactory type that could be intermediate to the segregated and uniform types already described.

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Identification of two distinct genes at the vertebrate TRPC2 locus and their characterisation in a marsupial and a monotreme.

BACKGROUND: The vomeronasal organ (VNO) detects pheromones via two large families of vomeronasal receptors: vomeronasal receptor 1 (V1R) and vomeronasal receptor 2 (V2R). Both VRs have a common receptor activation cascade involving transient receptor potential channel, subfamily C, member 2 (TRPC2). RESULTS: We characterised the TRPC2 locus in a marsupial, the tammar wallaby (Macropus eugenii), and identified two independently regulated genes not previously recognised as distinct. 3'-located exons comprise bona fide TRPC2 whilst 5'-located exons, previously identified as part of TRPC2, comprise a distinct gene, which we term XNDR (XRCC1 N-terminal domain-related). The two genes show contrasting expression patterns in the tammar: TRPC2 is specifically expressed in adult and developing VNO, whereas XNDR is widely expressed in many tissues suggesting a non-VNO-specific role. Strong expression of TRPC2 was detected only after about day 30 post-partum, suggesting that the VNO may not be functional during early pouch life of the tammar. Similarly restricted expression of TRPC2 and widespread expression of XNDR was also detected in the platypus. Bioinformatic analysis of the genomes of a wide range of species suggests that the identity of XNDR and TRPC2 as distinct genes is conserved among vertebrates. Finally, we analysed the promoter of mammalian TRPC2 and identified a conserved binding site for NHLH1, a transcription factor previously implicated in VNO receptor neuron development. CONCLUSIONS: Two functionally distinct vertebrate genes-XNDR and TRPC2 - occupy a genomic locus that was previously defined as a single gene in the mouse. The former is widely expressed with a putative role in DNA repair, while the latter shows VNO-specific expression under the probable regulation of NHLH1.

The effect of pregnant and oestrous females on male testosterone and behaviour in the tammar wallaby.

Tammar wallaby females (Macropus eugenii) are seasonally breeding marsupials with a post-partum oestrus after a highly synchronised birth period when testosterone concentrations rise in males. Chemical communication appears to be important for mating, as males show checking behaviour, sniffing the urogenital opening (UGO) and the pouch of females. This study investigates whether the presence of pregnant and oestrous females directly influences testosterone in males and if oestrous odours or secretion from the pouch or UGO are attractive. Concentrations of plasma testosterone were measured in males housed with pregnant and oestrous females during two consecutive cycles in the breeding season, and an artificially induced cycle in the non-breeding season. Males were also tested for their interest in swabs taken from the urogenital opening (UGO) or pouch of oestrous females. Testosterone increased sharply in males in the presence of pregnant and oestrous females during all cycles in both seasons, but there was no change when males were exposed to non-cycling females in lactational or seasonal diapause. Males had no preference for either oestrous or non-oestrous samples taken from the pouch or from the UGO from oestrous females. This study confirms that the increase in plasma testosterone in tammar males can be induced through the presence of pregnant and oestrous females, regardless of season and that the increase began when the females were in late-pregnancy. This confirms that the male's reproductive state is dependent on a signal from females and is not blocked through seasonal effects.

The olfactory system of the tammar wallaby is developed at birth and directs the neonate to its mother's pouch odours.

In kangaroos and wallabies at birth the highly altricial newborn young climbs unassisted from the urogenital opening to the teat. Negative geotropism is important for the initial climb to the pouch opening, but nothing is known of the signals that then direct the neonate downwards to the teat. Here we show that the newborn tammar wallaby (Macropus eugenii) has the olfactory apparatus to detect smell. Both the main olfactory system and vomeronasal organ (VNO) are developed at the time of birth. Receptor cells of the main olfactory epithelium immunopositive for G(oalpha)-protein project to the three layered main olfactory bulb (MOB). The receptor epithelium of the VNO contains G-protein immunopositive cells and has olfactory knob-like structures. The VNO is connected to an area between the two MOBs. Next, using a functional test, we show that neonates can respond to odours from their mother's pouch. When neonatal young are presented with a choice of a pouch-odour-soaked swab or a saline swab, they choose the swab with their mother's pouch secretions significantly more often (P<0.05) than the saline swab. We conclude that both olfactory systems are capable of receiving odour signals at birth, a function that must be a critical adaptation for the survival of an altricial marsupial neonate such as the tammar for its journey to the pouch.

The vomeronasal organ of the tammar wallaby.

The vomeronasal organ is the primary olfactory organ that detects sexual pheromones in mammals. We investigated the anatomy of the vomeronasal organ of the tammar wallaby (Macropus eugenii), a small macropodid marsupial. Pheromones may be important for activation of the hypothalamo-pituitary axis of tammar males at the start of the breeding season because plasma testosterone and luteinizing hormone concentration in males rise concurrently with pregnancy and the post-partum ovulation in females. The gross anatomy and the connection to the brain of the vomeronasal organ were examined by light and electron microscopy in adult male and female tammars. The vomeronasal organ was well developed in both sexes. The vomeronasal organ is a tubular organ connected at the rostral end via the nasopalatine duct (incisive duct) to the mouth and nasal cavity. At the rostral end the lumen of the vomeronasal organ was crescent shaped, changing to a narrow oval shape caudally. Glandular tissue associated with the vomeronasal organ increased towards the blind end of the organ. The tammar has the typical pattern of mammalian vomeronasal organs with electron-dense supporting cells and electron-lucent receptor cells. Microvilli were present on the surface of both epithelia while cilia were only found on the surface of the non-receptor epithelium. Some non-receptor epithelial cells appeared to secrete mucus into the vomeronasal organ lumen. The vomeronasal organ shows a high degree of structural conservation compared with eutherian mammals. The degree of vomeronasal organ development makes it likely that, as in other mammals, pheromones are important in the reproduction of the tammar.