The smelling of Hedione results in sex-differentiated human brain activity

Highlights

• All putative human pheromone receptors are expressed in the olfactory mucosa.

• Heterologously expressed VN1R1 can be activated by the odorous substance Hedione.

• Hedione induces neuronal activity in extra-olfactory brain areas.

• VN1R1 is involved in gender-specific hypothalamic activations.

Abstract

A large family of vomeronasal receptors recognizes pheromone cues in many animals including most amphibia, reptiles, rhodents, and other mammals. Humans possess five vomeronasal-type 1 receptor genes (VN1R1–VN1R5), which code for proteins that are functional in recombinant expression systems. We used two different recombinant expression systems and identified Hedione as a ligand for the putative human pheromone receptor VN1R1 expressed in the human olfactory mucosa. Following the ligand identification, we employed functional magnetic resonance imaging (fMRI) in healthy volunteers to characterize the in vivo action of the VN1R1 ligand Hedione. In comparison to a common floral odor (phenylethyl alcohol), Hedione exhibited significantly enhanced activation in limbic areas (amygdala, hippocampus) and elicited a sex-differentiated response in a hypothalamic region that is associated with hormonal release.

Utilizing a novel combination of methods, our results indicate that the putative human pheromone receptor VN1R1 is involved in extra-olfactory neuronal activations induced by the odorous substance Hedione. The activation of VN1R1 might play a role in gender-specific modulation of hormonal secretion in humans.

Introduction

“Social odors” or pheromones are defined as chemicals that are released from one animal and evoke a change in the behavior or hormone system of another animal of the same species (Karlson and Luscher, 1959). Human chemosensory communication is highly complex and controversial (Wysocki and Preti, 2004). The axilla is an origin of human body odor, and axillary sweat is the most likely source of human pheromones. The effects of smelling the bouquets of axillary sweat were examined extensively in recent years. Functional imaging approaches to human brain activity, such as positron emission tomography (PET) or magnetic resonance imaging (MRI), were a focal point in recent studies. The sniffing of human body odor induces the activation of different neuronal networks than common olfactory stimuli (Lundstrom et al., 2008). Further, information about anxiety and emotional stress are transmitted via axillary sweat. Neuronal activation patterns show the involvement of areas that are known for the processing of emotions and the regulation of empathy and attention. Therefore, the olfactory system likely mediates emotional contagion, although participants cannot consciously differentiate perceived chemosensory stimuli (Mujica-Parodi et al., 2009, Prehn-Kristensen et al., 2009). A study of human tears provides another example of social chemical communication. Men showed reduced activity in brain substrates of sexual arousal when sniffing women's tears induced by sadness (Gelstein et al., 2011). Derivatives of human sex hormones are discussed as single potent molecules that evoke physiological or behavioral responses. The steroids, 4,16-androstadien-3-one (AND) and estra-1,3,5(10),16-tetraen-3-ol (EST) are produced in a gender-specific pattern, and these steroids were linked to pheromone-like activities because they influence mood, physiological arousal, visual perception and brain activity (Grosser et al., 2000, Lundstrom et al., 2003, Bensafi et al., 2004, Villemure and Bushnell, 2007, Zhou et al., 2014).

There are two types of chemoreceptor families recognizing pheromones in rodents: ~ 240 vomeronasal-type 1 receptors (V1r) and ~ 61 vomeronasal-type 2 receptors (V2r). V1rs and V2rs are expressed in vomeronasal sensory neurons (VSNs) of the vomeronasal organ (VNO), which is a distinct structure besides the main olfactory epithelium (MOE) located in the nasal cavity (Dulac and Axel, 1995, Herrada and Dulac, 1997, Yang et al., 2005, Young et al., 2010). Isolated VSNs are activated by nonvolatile peptides of the main histocompatibility complex (MHC) class I, thought to be responsible for kin recognition alongside other volatile pheromone substances (Leinders-Zufall et al., 2000, Leinders-Zufall et al., 2004). Only one receptor — agonist pair in the vomeronasal system was identified so far, which showed that murine V1rb2 expression is required to elicit a response to the pheromone 2-heptanone (Boschat et al., 2002). V1r family sizes show species-specific expansions throughout mammalian evolution, and the functional repertoire roughly correlates with anatomical observations of VNO size and quality. V1r family size declines in primates, and the V1r repertoires of all Old World monkeys and apes consist primarily of pseudogenes (Young et al., 2010). There are over 100 functional V1rs in the rat and mouse genomes, but only five intact vomeronasal-type 1 receptor genes (VN1Rs) are found in human and chimpanzee genomes (Liman, 2006). At least one of the five intact VN1R genes is expressed in cells of the human olfactory mucosa (Rodriguez et al., 2000).

Humans also suffered inactivating mutations in the vomeronasal signal transduction gene Trpc2 (Liman and Innan, 2003). The loss of molecular components of VNO signaling is consistent with the absence of a functioning VNO in adult humans, but it does not necessarily indicate a loss of the sensing and functioning of “social odors” (Witt and Hummel, 2006). The detection of “social odors” was thought to be accomplished solely through the VNO, but it is now accepted that the main olfactory epithelium (OE) is also involved in the sensing of “social odors” (Brennan and Zufall, 2006, Frasnelli et al., 2011). In support of this view, surgical removal of the VNO in neonatal rabbits demonstrated that the stereotypic nipple search behavior occurred independently of the VNO via the main olfactory pathway (Distel and Hudson, 1985). Domestic pigs do not necessarily require the VNO for the detection of, and behavioral responses to, 5α-androst-16-en-3-one, which is a pheromone in boar's saliva (Dorries et al., 1997). Additionally, the olfactory receptor (OR) OR7D4 responded to AND and 5α-androst-16-en-3-one, a putative pheromone in humans, in a heterologous cell system. A common variant of OR7D4, which exhibits impaired function in vitro, correlated with variability in the perception of AND and 5α-androst-16-en-3-one in humans (Keller et al., 2007). Sensory neurons of the VNO that express the same vomeronasal receptors have neuronal projections to multiple glomeruli in the accessory olfactory bulb (AOB), which provides a direct pathway to hypothalamic areas where neuroendocrine levels can be regulated after pheromone detection (Li et al., 1990, Boehm et al., 2005). Receptors of the OE project to the main olfactory bulb (MOB). Individual mitral cells of the MOB in mice respond to volatile compounds in urine (Lin et al., 2005). Therefore, the MOB may also process social signals. Humans appear to lack a VNO and AOB (Brennan and Zufall, 2006, Frasnelli et al., 2011), but the smelling of AND causes hypothalamic activations that are gender-specific and similar to regions that control sexual orientation in other mammals (Savic et al., 2001, Savic et al., 2005, Savic, 2002, Berglund et al., 2006). If hypothalamic activations induced by AND are in fact gender-specific remains controversial (Burke et al., 2012).

The vomeronasal receptor VN1R1 is expressed in the human OE, and it is activated by volatile compounds in a heterologous system. However, the function of VN1R1 in vivo remains elusive (Shirokova et al., 2008). Our study indicates that not only VN1R1, but all intact VN1Rs are expressed in the human olfactory mucosa. We identified Hedione (HED) as a ligand for VN1R1 in two different heterologous expression systems and examined the pattern of cerebral processing in response to the smelling of HED in humans.