The role of the microbiome in the neurobiology of social behaviour

Microbes colonise all multicellular life, and the gut microbiome has been shown to influence a range of host physiological and behavioural phenotypes. One of the most intriguing and least understood of these influences lies in the domain of the microbiome's interactions with host social behaviour, with new evidence revealing that the gut microbiome makes important contributions to animal sociality. However, little is known about the biological processes through which the microbiome might influence host social behaviour. Here, we synthesise evidence of the gut microbiome's interactions with various aspects of host sociality, including sociability, social cognition, social stress, and autism. We discuss evidence of microbial associations with the most likely physiological mediators of animal social interaction. These include the structure and function of regions of the ‘social' brain (the amygdala, the prefrontal cortex, and the hippocampus) and the regulation of ‘social’ signalling molecules (glucocorticoids including corticosterone and cortisol, sex hormones including testosterone, oestrogens, and progestogens, neuropeptide hormones such as oxytocin and arginine vasopressin, and monoamine neurotransmitters such as serotonin and dopamine). We also discuss microbiome‐associated host genetic and epigenetic processes relevant to social behaviour. We then review research on microbial interactions with olfaction in insects and mammals, which contribute to social signalling and communication. Following these discussions, we examine evidence of microbial associations with emotion and social behaviour in humans, focussing on psychobiotic studies, microbe–depression correlations, early human development, autism, and issues of statistical power, replication, and causality. We analyse how the putative physiological mediators of the microbiome–sociality connection may be investigated, and discuss issues relating to the interpretation of results. We also suggest that other candidate molecules should be studied, insofar as they exert effects on social behaviour and are known to interact with the microbiome. Finally, we consider different models of the sequence of microbial effects on host physiological development, and how these may contribute to host social behaviour.     

The sense of smell: molecular basis of odorant recognition

Most animal species rely on odorant compounds to locate food, predators, or toxins. The sense of smell is also involved in animal communication, and revealing the underlying mechanisms will therefore facilitate a deeper understanding of animal behaviour. Since the 1940s different theories have speculated on the fundamental basis of olfaction. It was assumed that odorant molecules were recognized by selective protein receptors in the nose, triggering a nervous signal processed by the brain. The discovery of these receptors in the early 1990s allowed great progress in understanding the physiological and biochemical principles of olfaction. An overview of the different mechanisms involved in the coding of odour character as well as odour intensity is presented here, focusing on the biochemical basis of odorant recognition. Despite the enormous progress achieved in recent years, details of odorant-receptor interaction at the molecular level and the mechanisms of olfactory receptor activation are poorly understood. The likely role of metal ions in odorant recognition is discussed, and also the perireceptor events involved in odorant transport and biotransformation, with a view to providing a comprehensive overview of mammalian olfaction to guide future computational structural models and the design of functional experiments. Recent studies have analysed the olfactory genome of several species, providing information about the evolution of olfaction. The role of the olfactory system in animal communication is also described.     

Olfaction in Drosophila

The fruit fly, Drosophila melanogaster, is equipped with a sophisticated olfactory sensory system that permits it to recognize and discriminate hundreds of discrete odorants. The perception of these odorants is essential for the animal to identify relevant food sources and suitable sites for egg-laying. Advances in the last year have begun to define the molecular basis of this insect's discriminatory power. The identification of a large multi-gene family of candidate Drosophila odorant receptors suggests that, as in other animals, a multitude of distinct odorants is recognized by a diversity of ligand-binding receptors. How olfactory signals are transduced and interpreted by the brain remains an important question for future analysis. The availability of genetic tools and a complete genome sequence makes Drosophila a particularly attractive organism for studying the molecular basis of olfaction.     

Odorant-receptor interactions and odor percept: a chemical perspective

Receptor-ligand interaction models are generally based on a 'lock and key' concept. How far this holds true for olfactory receptors and odor molecules is currently uncertain. Here, we have investigated the response of a human olfactory receptor, OR1D2, to a broad array of odorants and found that there is no simple, direct correlation between a molecule's ability to activate this receptor and the odor impression elicited in the brain. In a parallel study on specific anosmia, we have found no evidence for odor-specific anosmia to either musk or amber, but rather to specific molecules within these categories. Cluster analysis confirmed that there is no simple correlation between molecular structure and impaired perception in either odor type. There are some differences in patterns of impairment between the two odor types and some evidence to suggest that subjects with specific anosmia to a given substance can identify its presence in a mixture. Taken together, our results show that simplistic 'lock and key' models of olfaction based on a concept of odor-quality-tuned receptors are inadequate, irrespective of the nature of the lock-key interaction. Receptor activation is only one step in a long chain of events leading from inhalation of odorants to perception of odor in the higher brain, and, therefore, although structure-odor correlations are useful tools for the design of novel odorants, caution should be exercised when extrapolating them to models of olfactory perception. Those seeking to understand the odorant-receptor interaction should use receptor activation rather than odor as input data.     

Signal recognition and chemoelectrical transduction in olfaction

The mimicking of olfaction is considered to be a promising approach for the construction of artificial odour-sensing systems. In the nose, the detection of volatile odorants begins when the odorant ligands interact with specific odorant receptors in the ciliary membrane of the olfactory neurons. A large family of genes encoding putative odorant receptors has been identified recently. Individual receptor types are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium. Ligand-receptor interaction triggers a rapid multistep reaction cascade, resulting in a “pulse” of second messengers that initiates an electrical response from the receptor neuron. Olfactory signalling is terminated by phosphorylation of receptors via a negative feedback reaction, catalyzed by specific kinases.     

Human olfactory receptor families and their odorants

The human nose detects volatile chemical stimuli by at least three different receptor families: odorant receptors, trace amine-associated receptors, and vomeronasal type-1 receptors. As G protein-coupled receptors, all of the few functionally characterized olfactory receptors share major functional features: when expressed in heterologous cell systems, they 1) respond to odorants of certain chemical groups, e.g., amines, aliphatic carboxylic acids or aldehydes, floral or fruity odorants, including certain key-food odorants, and putative pheromones, and 2) transduce their signals to intracellular cAMP signaling. However, little is known yet about specific differences in the functional designation of the three olfactory receptor families. Recently, two heterologous cell systems expressing olfactory signaling molecules have been developed. Different screening strategies will shed light on the yet sparsely available odorant specificity profiles and structure-function relationships of olfactory receptors, as well as the structure-activity relationships of their odorants.     

Expression of odorant receptor family, type 2 OR in the aquatic olfactory cavity of amphibian frog Xenopus tropicalis

Recent genome wide in silico analyses discovered a new family (type 2 or family H) of odorant receptors (ORs) in teleost fish and frogs. However, since there is no evidence of the expression of these novel OR genes in olfactory sensory neurons (OSN), it remains unknown if type 2 ORs (OR2) function as odorant receptors. In this study, we examined expression of OR2 genes in the frog Xenopus tropicalis. The overall gene expression pattern is highly complex and differs depending on the gene and developmental stage. RT-PCR analysis in larvae showed that all of the OR2η genes we identified were expressed in the peripheral olfactory system and some were detected in the brain and skin. Whole mount in situ hybridization of the larval olfactory cavity confirmed that at least two OR2η genes so far tested are expressed in the OSN. Because tadpoles are aquatic animals, OR2η genes are probably involved in aquatic olfaction. In adults, OR2η genes are expressed in the nose, brain, and testes to different degrees depending on the genes. OR2η expression in the olfactory system is restricted to the medium cavity, which participates in the detection of water-soluble odorants, suggesting that OR2ηs function as receptors for water-soluble odorants. Moreover, the fact that several OR2ηs are significantly expressed in non-olfactory organs suggests unknown roles in a range of biological processes other than putative odorant receptor functions.     

Expression patterns of two putative odorant-binding proteins in the olfactory organs of Drosophila melanogaster have different implications for their functions

The aqueous medium bathing the dendrites of olfactory neurons contains high concentrations of odorant-binding proteins (OBPs) whose role is still unclear. OBPs may facilitate interactions between odorants and their membrane-bound receptors, perhaps by increasing the water solubility of hydrophobic molecules. Alternatively, OBPs may be involved in the inactivation of odorants and other volatile molecules, preventing desensitization and/or protecting olfactory neurons from toxic chemicals. We report here novel features of the localization of two putative OBPs, PBPRP2 and PBPRP5, that have important and different implications for their role in olfaction. Unlike several other putative OBPs of Drosophila melanogaster that are only found in adult olfactory organs, PBPRP5 is also expressed in the larval olfactory organs, suggesting that it plays a common role in olfaction at both stages. In the adult, PBPRP5 expression is restricted to the sensillum lymph that bathes the olfactory dendrites of a subset of olfactory hairs, the basiconic sensilla. Since individual basiconic sensilla differ in olfactory specificity, PBPRP5 may be able to bind to and mediate olfactory responses to a wide range of odorants. In contrast, PBPRP2 is present in the space immediately below the antennal cuticle and in the outer cavity of approximately 30% of the double-walled coeloconic sensilla on the antennal surface. In neither case is PBPRP2 in contact with the dendritic membranes of olfactory neurons, making a carrier function unlikely for this protein. Instead, PBPRP2 may act as a sink, binding to odorants and other volatile chemicals and limiting their interactions with olfactory neurons.     

Effects of acidification on olfactory-mediated behaviour in freshwater and marine ecosystems: a synthesis

For many aquatic organisms, olfactory-mediated behaviour is essential to the maintenance of numerous fitness-enhancing activities, including foraging, reproduction and predator avoidance. Studies in both freshwater and marine ecosystems have demonstrated significant impacts of anthropogenic acidification on olfactory abilities of fish and macroinvertebrates, leading to impaired behavioural responses, with potentially far-reaching consequences to population dynamics and community structure. Whereas the ecological impacts of impaired olfactory-mediated behaviour may be similar between freshwater and marine ecosystems, the underlying mechanisms are quite distinct. In acidified freshwater, molecular change to chemical cues along with reduced olfaction sensitivity appear to be the primary causes of olfactory-mediated behavioural impairment. By contrast, experiments simulating future ocean acidification suggest that interference of high CO₂ with brain neurotransmitter function is the primary cause for olfactory-mediated behavioural impairment in fish. Different physico-chemical characteristics between marine and freshwater systems are probably responsible for these distinct mechanisms of impairment, which, under globally rising CO₂ levels, may lead to strikingly different consequences to olfaction. While fluctuations in pH may occur in both freshwater and marine ecosystems, marine habitat will remain alkaline despite future ocean acidification caused by globally rising CO₂ levels. In this synthesis, we argue that ecosystem-specific mechanisms affecting olfaction need to be considered for effective management and conservation practices.     

Olfactory detectability of L-amino acids in the European honeybee (Apis mellifera)

The honeybee is one of several insect model systems for the study of olfaction, yet our knowledge regarding the spectrum of odorants detectable by Apis mellifera is limited. One class of odorants that has never been tested so far are the amino acids, which are important constituents of floral nectar. Using the proboscis extension response paradigm, we assessed whether the odor of amino acids is detectable for honeybees and determined olfactory detection thresholds for those amino acids that were detectable. We found that honeybees are able to detect the odor of 5 of the 20 proteinogenic amino acids when presented at a concentration of 50 or 100 mM. Median olfactory detection thresholds for these 5 amino acids were 12.5 mM with L-tyrosine and L-cysteine, 50 mM with L-tryptophan and L-asparagine, and 100 mM with L-proline. All detection thresholds were much higher than reported concentrations of amino acids in floral nectars. We conclude that in the foraging and feeding context, honeybees are likely to detect amino acids through taste rather than olfaction. Across-species comparisons of the detectability of and sensitivity to amino acids suggest that the number of functional genes coding for olfactory receptors may affect both a species' sensitivity for odorants and the breadth of its spectrum of detectable odorants.     

Functional dissection of Odorant binding protein genes in Drosophila melanogaster

Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial.     

Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1

Animals in complex environments must discriminate between salient and uninformative sensory cues. Caenorhabditis elegans uses one pair of olfactory neurons called AWC to sense many different odorants, yet the animal can distinguish each odorant from the others in discrimination assays. We demonstrate that the transmembrane guanylyl cyclase ODR-1 is essential for responses to all AWC-sensed odorants. ODR-1 appears to be a shared signaling component downstream of odorant receptors. Overexpression of ODR-1 protein indicates that ODR-1 can influence odor discrimination and adaptation as well as olfaction. Adaptation to one odorant, butanone, is disrupted by ODR-1 overexpression. Olfactory discrimination is also disrupted by ODR-1 overexpression, probably by overproduction of the shared second messenger cGMP. We propose that AWC odorant signaling pathways are insulated to permit odor discrimination.     

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.     

Lake char (<Emphasis Type="Italic">Salvelinus namaycush</Emphasis>) olfactory neurons are highly sensitive and specific to bile acids

Bile acids have been implicated as chemical signals in spawning behaviour of lake char (Salvelinus namaycush). In this study, we investigated olfactory responses of lake char to bile acids by using the electro-olfactogram recording. Lake char detected 9 out of 38 bile acids tested at thresholds 0.02–0.5 nM. The most stimulatory included chenodeoxycholic acid, cholic acid, taurochenodeoxycholic acid, taurocholic acid, and taurolithocholic acid 3α-sulphate. Structure–activity analysis indicated that substituents in the side chain or hydroxyl sulphation were determinant elements for the recognition of individual bile acid receptors, while the position and orientation of hydroxyls or the type of amidation were important for effective stimulation. Three distinct types of concentration–response relationships were found, representing free, taurine- or glycine-amidated, and 3α-sulphated bile acids. Cross-adaptation and binary mixture experiments revealed the presence of multiple olfactory receptors for bile acids. Lake char were also capable of detecting petromyzonol sulphate at 1 nM, possibly via its own receptors. Our study further showed that the olfactory responses to bile acids were independent of those of known odorants including amino acids, prostaglandins and gonadal steroids. We conclude that lake char possess multiple olfactory receptors capable of discriminating bile acids produced and released by conspecifics.     

Olfactory receptor gene repertoires in mammals

In mammals, olfaction is mediated by two distinct organs that are located in the nasal cavity: the main olfactory epithelium (MOE) that binds volatile odorants is responsible for the conscious perception of odors, and the vomeronasal organ (VNO) that binds pheromones is responsible for various behavioral and neuroendocrine responses between individuals of a same species. Odorants and pheromones bind to seven transmembrane domain G-protein-coupled receptors that permit signal transduction. These receptors are encoded by large multigene families that evolved in mammal species in function of specific olfactory needs.     

After oxidation,zinc nanoparticles lose their ability to enhance responses to odorants

Electrical responses of olfactory sensory neurons to odorants were examined in the presence of zinc nanoparticles of various sizes and degrees of oxidation. The zinc nanoparticles were prepared by the underwater electrical discharge method and analyzed by atomic force microscopy and X-ray photoelectron spectroscopy. Small (1.2 ± 0.3 nm) zinc nanoparticles significantly enhanced electrical responses of olfactory neurons to odorants. After oxidation, however, these small zinc nanoparticles were no longer capable of enhancing olfactory responses. Larger zinc oxide nanoparticles (15 nm and 70 nm) also did not modulate responses to odorants. Neither zinc nor zinc oxide nanoparticles produced olfactory responses when added without odorants. The enhancement of odorant responses by small zinc nanoparticles was explained by the creation of olfactory receptor dimers initiated by small zinc nanoparticles. The results of this work will clarify the mechanisms for the initial events in olfaction, as well as to provide new ways to alleviate anosmia related to the loss of olfactory receptors.     

昆虫感觉气味的细胞与分子机制研究进展

昆虫作为地球上最为成功的类群,已经成功地进化了精细的化学感受系统,通过化学感受系统适应各种复杂的环境,保持种群的繁荣。自1991年在动物中发现嗅觉受体基因以来,关于昆虫感受化学信息的周缘神经系统的分子和细胞机制方面的进展十分迅速。文章主要就昆虫周缘神经系统的感受化学信息的分子和细胞机制进行综述。首先对昆虫感觉气味的细胞机制的研究进展进行简要介绍。昆虫嗅觉神经元在感受化学信息过程中起着极为重要的作用,昆虫嗅觉神经元上表达的嗅觉受体不同而执行着各异的功能。各种嗅觉神经元对于化学信息的感受谱有较大的区别;嗅觉神经元对化学信息类型、浓度、流动动态等产生相应的电生理特征反应。研究表明同一种神经原可以感受多种化学信息,而一种化学信息也可以被多种神经原所感受。由神经原对化学信息感受所形成的特征组合就是感受化学信息的编码。其次较为详细地论述与昆虫感受气味分子相关的一些蛋白质的研究进展。气味分子结合蛋白是一类分子量较小、水溶性的蛋白,主要位于化学感受器神经原树突周围的淋巴液中。在结构上的主要特征是具有6个保守的半光氨酸和由6个α螺旋组成的结合腔。自1981年发现以来,已经在40余种昆虫中发现上百种。由于研究手段的不断进步,已经对该类蛋白的表达特征、结合特性以及三维结构和结合位点进行了大量的研究,提出了多个可能的功能假说,在诸多的假说中,较为广泛接受的是气味分子结合蛋白在昆虫感觉气味的过程中,是与疏水性的气味分子相结合,并将气味分子运输到嗅觉神经原树突膜上的嗅觉受体上。这些处于树突膜上的嗅觉受体则是昆虫感觉气味过程中的另一个十分重要的蛋白质。目前,已经在果蝇、按蚊、蜜蜂和家蚕等10余个昆虫种类中发现上百个嗅觉受体蛋白基因。这类蛋白是跨膜蛋白,一般具有7个跨膜区,整个蛋白的氨基酸残基在400～600个。昆虫的嗅觉受体蛋白的N-端在胞内,而C-端在胞外,这与G耦联蛋白不同。而且,昆虫的一个嗅觉神经元可以表达1～3个嗅觉受体蛋白,也与哺乳动物的一个神经元只表达一种受体蛋白有所不同。每种嗅觉受体可以感受多种气味分子,而一种气味分子可以被多个嗅觉受体所感知,这样组成了感受化学信息的编码谱。最近采用基因敲除技术和膜片钳技术研究发现,昆虫的嗅觉受体蛋白在信号传导中也有特殊性,即嗅觉受体可以直接作为离子通道,而引起动作电位。还有近来的研究表明,神经膜蛋白对于果蝇的性信息素感受神经元感受性信息素cVA是必要的。实际上,昆虫对于化学信息的感受和信号的转导,并不是上述蛋白单独起作用完成的,而是多种蛋白相互作用的结果。论文最后对该领域研究内容进行了展望。     

A screen for genes expressed in the olfactory organs of Drosophila melanogaster identifies genes involved in olfactory behaviour

Background

For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system.

Methodology/Principal Findings

We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions.

Conclusions/Significance

We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms.