Can pheromones be received other than by odor?

Can pheromones be received other than by odor?

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I'm trying to understand how deodorants affect pheromones in humans.

Wiki states:


In humans, the formation of body odor happens mostly in the axillary region. These odorant substances serve as pheromones which play a role related to mating.


A deodorant is a substance applied to the body to prevent body odor caused by the bacterial breakdown of perspiration in armpits, feet, and other areas of the body.

Can the opposite sex receive pheromones if a person uses crystal deodorant (doesn't have a fragrance; doesn't block sweating; prevents body odor by creating an invisible, protective barrier against odor-causing bacteria)?

We have only 5 senses, a 6th sense has been controversial, and some 6th senses are sometimes reported in new research in newspapers, but none have been conclusively been added to the clearly known 5. There is a second olfactory organ that deals with social signals in other animals, that we possess, the vomeronasal organ.

Pheromones are only olfactory molecules, so you can't get them other than by catching molecules and analysing them, i.e. smelling.

Humans react very strongly by attraction to some perfumes other than pheremones, and you arent supposed to block all your smells, only make them containable, a bit is probably a good thing.

Scientists did publish a study claiming that people can tell if they make a good immune response / metabolic genetic combo with a partner from smelling a potential partner's t-shirt.

Sniffing Pheromones

Smell is important to allow us to taste our food, and can even trigger memories. But in some animals, special molecules called pheromones can also trigger certain types of behaviour, such as mating. Kate Lamble spoke to Greg Jefferis from the Cambridge Medical Research Council Laboratory of Molecular Biology, or the MRC LMB, investigates the pathways between smell and behaviour in the fruit fly

Greg - A pheromone is, I guess, a specialised smell molecule that's used to communicate between individuals of a species. So, it's produced by one member of a species and used to signal for example to a member of the opposite sex, is one of the classic cases, so sex pheromones. So, there's normally a specialised detection process as well and we're particularly interested in what happens within the brain when those odours are detected.

Kate - If we looked at the pheromones, would it look any different from a normal smell molecule?

Greg - No, not particularly and in fact, there are some pheromones which can be detected by our main olfactory epithelium, so the nose, that can have very specific meanings for certain species.

Kate - So, if they don't look any different, I suppose it's about how it is received. How does smelling a pheromone get translated into behaviour in animals?

Greg - So, like any odour, the first thing is that this small molecule needs to diffuse up to a receptor at the top of your nose and actually bind to a receptor there. And one of the key advantages of using insects is that these receptors and molecules have been identified for some time. So, once it binds to the receptor it then makes the neuron on which that receptor sits electrically active and so, you've turned this chemical binding into an electrical signal which can then talk to the rest of the brain and trigger behaviour responses.

Kate - And you study this in fruit flies. Why are fruit flies such a great animal to look at this in?

Greg - So, various reasons. There are lots of, people who have been studying fruit flies for years. Obviously, there are all sorts of powerful tools. There's also an issue of complexity. Fruit flies have only 50 receptor genes whereas a mouse has 1300 olfactory receptor genes. So, it's a bit easier to find the receptor for a particular smell in a fruit fly than a mouse. Also, once you found the receptor, it's a lot easier to do experiment in a fly to try and figure out what the fly's brain is doing with this information and that's really the kind of work that my lab is doing.

Kate - So, what pheromones do fruit flies react to?

Greg - So, the one that's been best studied is a pheromone called cva cis-Vaccenyl Acetate. So, this is a molecule that's produced by male flies and signals to both males and females. So, it seems to be attractive for females. It makes them more ready to mate with the male, but it's repulsive for males. It actually makes them more aggressive, more likely to fight with each other. So, this is interesting, right? It's the same molecule but different effects on the two sexes.

Kate - That is interesting. We've got an email in from Teo Gibson who asks us, "Can humans smell pheromones or is it just a myth?"

Greg - I think it partly depends on your definition of pheromone. So, I think one of the big points at least classically has been that the pheromone should be a molecule that has some kind of unconditioned response. That is that the first time you smell it, it's going to make you produce some kind of behaviour and you're always going to produce that behaviour when you smell the pheromone. Now of course, we expect most things to be highly contextual especially in humans, there are lots of signals that interact and also, that there's lots of learning. Most of the sort of work on human pheromones has found it very hard to tease apart sort of learned associations from something that might be innate as it were.

Kate - I can imagine with a human, so much of will comes into it as well, that a fruit fly would automatically respond to something whereas we're able to stop ourselves to a certain extent. Teo goes on to say, she read an article a while back about a woman judging a man's breed worthiness based on the smell of a sweaty shirt. Is that true?

Greg - So, maybe we should bring in Darren (Logan from the Wellcome Trust Sanger Institute) here. I think he's got lots of experience at least in dealing with that kind of question, if not with smelling smelly t-shirts, but.

Darren - So, it is true that the study was done that showed that people prefer the smell of people who are unrelated to them at certain of their menstrual cycle, a woman does. This was classically done by making people wear no toiletries and wearing a white t-shirt and sleeping in that t-shirt and then sniffing it. It's not the most pleasant of experiments. So, there's quite a long history of these types of experiments. Indeed, there appears to be some sort of effect. Quite how it works and what people are smelling, we don't yet know.

Ginny Smith - Now Darren I think you told me earlier that you actually have an example of a human pheromone with you. Is that right?

Darren - So this is a chemical called androstenone and it's first identified in the saliva of male pigs and it drives female pigs wild. Subsequently, it was found in male sweat and so, it was studied because of its pheromonal properties in a pig as a putative human pheromone. What's really interesting about it is it smells very differently to different people. So, if you'd like to have a sniff and tell me if you can smell it.

Kate - This is one of those quizzes that tells you about your personality. You don't want to get it wrong

Darren - So, a lot of people can't smell it at all.

Kate - I can smell something. It's not driving me wild. It's quite sweet, but quite subtle to me.

Darren - So, that's interesting. What about you Ginny?

Kate - He's definitely worked something out about my personality that I don't want to be revealed on air.

Ginny - I'm not smelling anything.

Kate - You can't smell anything.

Darren - So, most people describe this as urinous or sickening or sweaty in some way. But a small proportion of people describe it as sweet and a lot of people also can smell it. I can't smell it, and so we have one person that can smell it that quite likes it. I think the other interesting thing about this chemical is irrespective of its potential pheromonal properties is that it is perhaps the most variable odour that has the most variable responses to smell to it. So, whether it's a pheromone, the jury is out I would say, but it certainly is an interesting smell.

Sex smells: pheromones in humans

By the middle of the 20th century, biologists had become aware of a unique type of communication occurring among insects. The communication involved the secretion of substances that were similar to hormones in some ways, but also very different. While hormones are secreted into the bloodstream to elicit some reaction in the body, these newly identified substances exited the body and were used to elicit a reaction in a conspecific (another organism of the same species). They were given the name pheromones, from the Greek pherin (to transfer) and hormon (to excite).

Since that time, a number of pheromones have been identified in invertebrates and vertebrates alike. For example, when a honeybee hive is disturbed, guard bees produce a pheromone that alerts other bees in the hive it encourages them to exit the hive and promotes aggressiveness. Beekeepers know all about this signal they use smoke to calm an angry hive of bees because smoke inhibits receptors in bee antennae that detect the pheromone.

Pheromones that elicit a variety of behaviors have been identified in a number of species. Some pheromones, like those seen in bees, are used to call conspecifics together to attack or defend something, some aid in marking territory, and others leave trails that conspecifics can follow (to a stash of food, for example). Many pheromones also are associated with sex.

A large number of species, from microorganisms on up, release pheromones that play some role in mating. Some species of bacteria use pheromones to tell other bacteria to prepare to receive a transfer of genetic material in a very unromantic form of bacterial "sex" called conjugation. Even mammals, however, communicate with sex pheromones. Males of a number of species investigate the anogenital region of females with their nose, which contains a special pheromone-detector called the vomeronasal organ. Through exposure to pheromones, the male may be able to tell if the female is ovulating and will be receptive to his advances.

Although pheromones have been detected in many species, it has long been debated if they play any role in human communication. One reason that some have argued against an important role for pheromones in humans is that evidence suggests we don't have a functional vomeronasal organ. However, there do seem to be some examples of hidden chemical signaling between people.

Perhaps the best known putative pheromone mechanism in humans is the McClintock effect. The McClintock effect describes menstrual synchrony, which is when the menstrual cycles of women living in proximity to one another begin to synchronize, or start at around the same time. The effect was named after psychologist Martha McClintock, who hypothesized that pheromones were responsible for the synchronization.

The McClintock effect is controversial, and some argue that it isn't a real biological effect much less something caused by pheromones. But a number of other studies have also found indications of possible human pheromonal communication. From these studies, two steroids in particular have emerged as potential pheromones: androstadienone and estratetraenol.

Androstadienone, a metabolite of testosterone, is found in male semen and in secretions from the armpit area. Research has suggested it may promote physiological arousal in women, but not in heterosexual men. Estratetraenol, on the other hand, is an estrogen found in female urine. Estratetraenol has been found to affect autonomic arousal in men. Thus, some studies suggest (though this is still a controversial area) that androstadienone and estratetraenol are pheromones that contain some information detectable by the opposite sex.

A point-light walker used to demonstrate human gait.

A study to be published this month in Current Biology investigated information about gender that might be conveyed by these two putative pheromones. The investigators, Zhou et al., explored the effects of androstadienone and estratetraenol on the attribution of gender to point-light walkers (PLWs) displayed on a screen. PLWs are a collection of dots that represent human movement (see gif to the right). By changing settings, PLWs can adopt a more masculine or more feminine gait.

Zhou et al. exposed heterosexual and homosexual or bisexual men and women to either androstadienone, estratetraenol, or a control solution while viewing PLWs that displayed a spectrum of walking styles that ranged from a feminine gait to a masculine gait, with gender-neutral gaits falling in between the two. The participants, after watching the PLW walk very briefly, had to make a judgment as to whether the figure was masculine or feminine.

The researchers found that exposing heterosexual males to estratetraenol decreased the frequency of "male" responses, but it didn't affect ratings from heterosexual females. Exposing heterosexual females to androstadienone increased the frequency of "male" responses, but this didn't occur in heterosexual males. The results from the homosexual and bisexual groups were a little more ambiguous estratetraenol didn't have an effect and androstadienone increased "male" responses in homosexual men but only to a gender-neutral PLW (and even then it was barely statistically significant).

Zhou et al. hypothesize that estratetraenol and androstadienone were biasing men and women, respectively, to discern the opposite sex in the movement of the PLWs. Thus, the authors argue that these substances convey information about masculinity and femininity. If true, what this means for everyday male-female interactions is unclear. The concentrations of the steroids that Zhou et al. used were much higher than you'd be exposed to just by standing next to someone on the subway.

So, there is much still to be learned about human pheromones. Even if androstadienone and estratetraenol are capable of communicating gender-specific information, their actual effect on human behavior today may be negligible. Thus, human pheromones may just be vestigial artifacts from our evolutionary history that we don't really have a use for anymore. On the other hand, there may be a complex system of communication occurring between people all the time that we are completely unaware of. And this system of communication could be shaping important decisions in your life, such as who you mate with, without your conscious realization.

A Closer Look: Tarsal Glands / Footprint Pheromone

When honey bees walk across a surface, their feet often deposit an attractive, oily, colorless secretion that has a low volatility. This secretion has been shown to affect the behavior of other workers, thus is considered to be a pheromone. This chemical has been termed “footprint pheromone’ or “trail pheromone.” The secretion is believed to originate from the tarsal (Arnhart) glands. These glands are located in the 5th tarsomere of the fore-, mid- and hind-legs of adult honey bee queens, workers and drones. Their structural features are not caste or sex specific. The tarsal glands have the shape of a flattened sac within each of the last tarsal segments of each leg (Lensky et al. 1985). Each gland consists of a unicellular layer which surrounds and secretes into a sac-like cavity which forms the reservoir of the gland. The unicellular layer of epithelial cells contain an abundance of cellular organelles consistent with a secretory activity (Goodman 2003). The chemical is deposited by the terminal arolium between the tarsal claws as the bee walks about. In addition to the feet, it is deposited by the tip of the abdomen, which often trails over the surface as the bee walks (Caron and Connor 2013).

The cells of the tarsal gland differ from those of the Nasonov gland in that they do not have ducts to transport the secretory material either to the reservoir or to the exterior. The gland cells are bounded by cuticle and to enter the reservoir the secretion must cross this barrier (Goodman 2003). This type of gland cell is classified as class 1 (Noirot et al. 1974). It is not clear how the secretory material crosses the cuticle – does it diffuse through the cuticle or make use of a pore canal system? The means by which the secretion exits the tarsal gland cells and gets to the reservoir remains to be determined.

Honey bees show a variety of “footprint chemicals” which have been characterized by chromatographic techniques. Included are alkanes, alkenes, alcohols, organic acids, ethers, esters and aldehydes. Tarsal glands secrete 12 compounds specific to queens, 11 specific for workers and one specific to drones (Lensky et al. 1987). A further difference lies in their rate of secretion, the tarsal gland of the queen secreting at a much higher rate than that of the worker and the drone. The secretion of six-month-old queens is greater than two-year-old queens the rate of secretion of the workers’ glands is 10-15 times less than that of the queen’s (Lensky et al. 1984).

Lateral view of end of last tarsal joint of first foor (Tar) showing empodium in ordinary position when not in use.

Secretions with very different functions are deposited by the tarsi of both queens and workers (Blum 1992). In the worker, the pheromone appears to belong to those chemicals that assist the bee in orientation. Workers deposit a persistent trail pheromone at their hive entrance and the attractiveness of this secretion increases with the number of workers depositing it (Butler et al. 1970). It appears that bees also mark forage sites with the footprint pheromone, thus increasing their attractiveness to other foragers (Ferguson and Free 1979). Thus flowers and sites containing artificial pheromone lures are more attractive to other workers than similar resources that have not been marked with footprint pheromone. There is also some suggestion that footprint pheromone may help short-term marking of individual flowers whose nectar source has been depleted – helping to avoid unproductive visits (Free 1987).

The queen leaves her special mark.

Butler et al. (1969) showed that crawling workers, apparently involuntarily, deposit a ‘footprint substance’ that attracts other workers and stimulates them to enter the hive. Homecoming honey bees are also attracted by an odor in the hive atmosphere which may be part of the ‘footprint substance.’ This footprint pheromone, which is certainly perceived olfactorily and possibly also chemotactically, is persistent but probably not colony specific. Glass entrance tunnels that have been marked with this trail pheromone are much preferred by homecoming bees to clean glass entrance tunnels. The attractiveness of an entrance tunnel increased with the number of workers that had previously used it up to about 400 workers, thereafter its attractiveness failed to increase further. The trail pheromone of workers from another colony was also attractive but slightly less so than that of workers from a bee’s own colony.

The floor and inside walls of the hive or nest and the combs themselves are also probably marked with trail pheromone. The accumulation of trail pheromone on comb may partially explain why old comb is more attractive than new (Free 1987). Butler (1967) found that bees he had trained to forage in a darkened arena produced an odor trail between their hive and the dish of sugar syrup.

It is well established that a glass dish on which bees have been foraging for sucrose syrup is more attractive to potential foragers than a clean dish, probably because of an attractive trail pheromone the foraging bees have left behind. Experiments that have demonstrated this involved training bees to collect sugar syrup from tubes or dishes placed on a circular table. The tubes or dishes with syrup were replaced by empty ones, provided with different odors and placed equidistant from the table center the number of bees that landed on or touched each tube or dish was compared. The table was rotated continuously so the bees did not become conditioned to any particular position (Ribbands 1954 Butler et al. 1969 Ferguson and Free 1979).

Bees visiting a site, mark it with an attractive pheromone irrespective of whether they have foraged successfully there or not (Free 1987). Ribbands (1954) showed that it was only necessary for a bee to land briefly on a particular empty tube for it to prefer that tube subsequently, Free (1970) found that would-be foragers were attracted to the odor bees had left on a glass sheet covering model flowers from which they could not obtain food and Ferguson and Free (1979) demonstrated that dishes on which bees had landed and had not foraged became attractive to others.

It seems that foraging bees may also have a preference for trail odor deposited by bees of their own colony. Bees from two colonies were trained to two separate but adjacent dishes of dilute sucrose syrup the dilute syrup in each dish was then replaced by concentrated syrup so that dancing and recruiting were encouraged. Newcomers that arrived were preferentially attracted to the dish visited by their nestmates (Kalmus and Ribbands 1952) and so deposition of a trail pheromone at a source of forage favors survival of their own colony.

Left hind leg of queen,
anterior or outer view.

It has been suggested that the tarsal gland secretion of the queen plays a part in inhibiting the construction of queen cups and hence in inhibiting queen rearing and swarming. Experiments were conducted to determine the role of population density of queen-right colonies and that of the queen bee pheromonal secretions, on the induction and inhibition of swarming queen cup construction during swarming and non-swarming seasons. Construction of queen cups was induced experimentally in overcrowded queenright colonies, during Winter, which is a non-swarming season. This construction was induced by a high population density of workers: above a threshold of 2.3 workers/ml there was a relationship between the number of cups constructed and the colony density. During the swarming season a relationship was established between the free volume of a hive (population density) and the number of queen cups constructed: 1.5 cups in a colony that occupied 80,960 ml, compared with 77 cups in a colony hived within a volume of 20,240 ml. Observations of the queen’s movements upon combs in colonies of high and normal population densities showed that in an overcrowded colony the queen bee was almost absent from the bottom edges of the comb, where queen swarming cups and cells are constructed. The glandular oily secretion from the queen’s tarsal glands is deposited by the foot-pads upon the comb surface. The rate of secretion by the queen’s tarsal glands was about 13 times higher than those of the workers. A bioassay based on increasing worker population densities for testing the inhibitory effects of the queen’s glandular extracts on the construction of queen cups was developed (Lensky and Slabezki 1981).

Footprint pheromone is everywhere in a hive.

The application of tarsal and mandibular glands’ secretion to comb bottom edges in overcrowded colonies (bioassay) caused the inhibition of queen cup construction. None of these two secretions affected construction of these cups when applied separately. They believe that due to colony overcrowding the queen bee is unable to deposit the non-volatile secretions from tarsal and mandibular glands along the comb edges and that the deficiency of the foot-print and mandibular pheromone triggers the construction of swarming cups along the non-inhibited areas (Lensky and Slabezki 1981).

Not all researchers agree that the tarsal glands are the site of production of the footprint pheromone (Winston 1987). Ferguson and Free (1979) reported that the odors of the head, thorax and abdomen are very active in inducing landing by workers searching for food. Thus, it is possible that this pheromone, while it is deposited by the feet, originates elsewhere on the body (Butler et al. 1969).

The footprint pheromone is capable of inducing disoriented workers to expose their Nasonov glands (Ferguson and Free 1981). Thus, this pheromone can work in concert with the Nasonov scent to aid workers which have become temporarily disoriented in the vicinity of the hive entrance. The attractiveness of synthetic Nasonov pheromone on the recruitment of foragers to glass dishes of sugar water has been compared with that of footprint pheromone (Williams et al. 1981). Dishes marked with footprint pheromone were visited more than clean dishes, while dishes marked with either footprint pheromone or synthetic Nasonov pheromone were equally attractive. However, dishes marked with both pheromones received a greater frequency of visits than any others. These experiments show that the footprint pheromone enhances the attractiveness of the Nasonov pheromone to foragers trained to visit a particular site for food.

Blum, M.S. 1992. Honey bee pheromones. In: Graham, J.M. (ed). The Hive And The Honey Bee, Dadant & Sons, Hamilton, IL, pp. 373-394.
Butler, C.G. 1967. Insect pheromones. Biol. Res. 42: 42-87.
Butler, C.G., D. J. C. Fletcher and D. Watler 1969. Nest-entrance marking with pheromones by the honeybee- Apis mellifera L., and by a wasp, Vespula vulgaris L. Anim. Behav. 17: 142-147.
Butler, C.G., D.J.C. Fletcher and D. Watler 1970. Hive entrance finding by honeybee (Apis mellifera) foragers. Anim. Behav. 18: 78-91.
Caron, D.M. and L.J. Connor 2013. Honey Bee Biology And Beekeeping. Wicwas Press, Kalamazoo, MI, 368 pp.
Ferguson, A.W. and J.B. Free 1979. Production of a forage-marking pheromone by the honeybee. J. Apic. Res. 18: 128-135.
Ferguson, A.W. and J. B. Free 1981. Factors determining the release of Nasonov pheromone by honeybees at the nest entrance. Physiol. Ent. 6: 15-19.
Free, J.B. 1970. Effect of flower shapes and nectar guides on the behaviour of foraging honeybees. Behaviour. 37: 269-285.
Free, J.B. 1987. Pheromones of Social Bees. Comstock Publishing Associates, Ithaca, NY, 218 pp.
Goodman, L. 2003. Form and Function in the Honey Bee. International Bee Research Association, Cardiff, UK, 220 pp.
Kalmus, H. and C.R. Ribbands 1952. The origin of the odours by which honeybees distinguish their companions. Proc. Royal Soc. B. 140: 50-59.
Lensky, Y. and Y. Slabezki 1981. The inhibiting effect of the queen bee (Apis mellifera L.) foot-print pheromone on the construction of swarming queen cups. J. Insect Physiol. 27: 313-323.
Lensky, Y., P. Cassier, A. Finkel, A. Teeshbee, R. Schlesinger, C. Delorme-Joulie and M. Levinsohn 1984. The tarsal gland of the honeybee (Apis mellifera L.) queens, workers and drones, biological effects. Ann. Sci. Nat. Zool. 6: 167-175.
Lensky, Y., P. Cassier, A. Finkel, C. Delorme-Joulie and M. Levinsohn 1985. The fine structure of the tarsal glands of the honeybee Apis mellifera L. (Hymenoptera). Cell Tissue Res. 240: 153-158.
Lensky, Y., A. Finkel, P. Cassier, A. Teeshbee and R. Schlesinger 1987. The tarsal glands of honeybee (Apis mellifera L.) queens, workers and drones—chemical characterization of footprint secretions. Honeybee Sci. 8: 97-102.
Noirot, C., A. Quennedey and R. F. Smith 1974. Fine structure of insect epidermal glands. Ann. Rev. Entomol. 19: 61-81.
Ribbands, C.R. 1954. Communication between honeybees. 1. The response of crop-attached bees to the scent of their crop. Proc. Royal Entomol. Soc., London. 29: 141-144.
Williams, I.H., J.A. Pickett and A.P. Martin 1981. Nasonov pheromone of the honey bee Apis mellifera L. (Hymenoptera: Apidae) Part II. Bioassay of the components using foragers. J. Chem. Ecol. 7: 225-237.
Winston, M.L. 1987. The Biology Of The Honey Bee. Harvard University Press, Cambridge, MA, 281 pp.

Clarence Collison is an Emeritus Professor of Entomology and Department Head Emeritus of Entomology and Plant Pathology at Mississippi State University, Mississippi State, MS.

Part 2: Molecular Biology of Pheromone Perception

00:00:06.04 So, what's happening specifically regarding the detection of pheromones?
00:00:14.18 What are the systems that detect pheromones, how is this information processed
00:00:20.27 within the brain and how are behaviors, specific behaviors, generated?
00:00:26.12 How does an animal know that the signal comes and leads to fighting behavior
00:00:31.24 or mating behavior? How is the quality of the pheromonal information received,
00:00:38.14 perceived and how does it lead to behavioral changes?
00:00:43.01 As I mentioned just before, there are these two systems,
00:00:50.05 the vomeronasal system on one hand, the olfactory system on the other hand,
00:00:54.19 and the assumption for a very long time
00:00:58.15 was that the vomeronasal system was specialized into the detection of pheromones
00:01:06.03 and the olfactory system was specialized in the detection of odorant chemicals.
00:01:11.16 And this notion came from surgical ablation experiments
00:01:20.03 in which people had surgically ablated the olfactory epithelium
00:01:24.07 and this led to impairment of odorant detection,
00:01:28.14 or surgical ablation of the vomeronasal organ and this led to defect in mating or aggressive behavior
00:01:36.04 and therefore, presumably the detection of pheromones.
00:01:39.16 So, surgical experiments, surgical ablation, were a big cue
00:01:44.10 into the role of each of these two separate systems
00:01:47.29 and also the notion that these two systems share the work
00:01:52.25 between cognitive smell and instinctive smell
00:01:55.21 also originates from the study of the central projection of this system.
00:02:01.25 So, the olfactory epithelium is connected to the main olfactory bulb,
00:02:07.16 and in turn, to a number of nuclei in the brain
00:02:11.26 that are together forming what is called the primary olfactory cortex
00:02:18.18 and then the information is distributed very widely within cortical and neocortical areas of the brain
00:02:24.16 and therefore leads to this cognitive perception of a smell.
00:02:29.05 And in contrast, information that is detected by the vomeronasal organ
00:02:35.08 seems to be processed by an entirely different and independent central pathway
00:02:41.08 from the vomeronasal organ to accessory olfactory bulb
00:02:45.15 to then specific areas of the medial amygdala in the limbic system
00:02:50.23 that are themselves connected to specific areas of the hypothalamus
00:02:55.05 that are specialized in aggressive, and triggering aggressive and mating behaviors.
00:03:01.15 So, specialized reproduction and social behavior in general.
00:03:05.09 So, it seems, therefore, to make sense that these areas that are involve
00:03:10.26 the primary olfactory cortex and then higher cortical areas
00:03:14.23 would indeed be responsible for the cognitive detection of smell
00:03:20.08 whereas areas of the brain that are part more of the limbic system,
00:03:25.14 the amygdala and the hypothalamus, are more involved in processing pheromonal signals
00:03:32.05 and the trigger of reproductive and aggressive behavior.
00:03:35.25 So, this seems all very logical, and at the molecular level,
00:03:41.07 it was also very interesting to discover that neurons of the main olfactory epithelium
00:03:48.01 seem, through a set of channels that are cyclic nucleotide gated,
00:03:54.03 therefore, the signal transduction of olfactory signals use cyclic nucleotides
00:03:58.28 that in turn lead to the opening of ion channels
00:04:02.14 and therefore, enable the translation of the binding of odorant to the receptor
00:04:07.08 into an electrical signal, a change in membrane potential.
00:04:11.01 And in contrast, in the vomeronasal organ,
00:04:14.06 we don't find any functional cyclic nucleotide gated channels,
00:04:19.05 what we found several years ago in collaboration with Emily Liman and David Corey,
00:04:25.12 is the very strong and specific expression of a distinct ion channel called TRPC2,
00:04:31.23 that is again very highly and specifically expressed in vomeronasal organ
00:04:36.19 and is responsible for the VNO signal transduction.
00:04:40.19 So, at the molecular level we have these two ion channels
00:04:45.18 that are each essential for olfactory transduction and vomeronasal transduction
00:04:50.26 and these therefore provide a terrific genetic tools to investigate,
00:04:56.17 or reinvestigate, if you wish, the function of each of these two sensory pathways in the brain.
00:05:03.19 So, by genetic manipulation of the gene encoding the TRPC2 channel,
00:05:08.28 we perform a knockout of the TRPC2 channel and therefore, led to an animal,
00:05:15.12 generated a line of genetically modified mouse, in which the VNO does not function,
00:05:21.19 because the TRPC2 channel is non-functional, is mutated,
00:05:25.09 and therefore the entire vomeronasal pathway is made non-functional.
00:05:30.12 And therefore, this mutated animal, this mutant,
00:05:33.07 doesn't have a functional vomeronasal organ, is unable to detect pheromones,
00:05:37.22 and we can therefore investigate the physiological role of the vomeronasal organ
00:05:43.01 in the animal physiology and behavior.
00:05:47.24 And similarly, but looking at the knockout of the cyclic-nucleotide gated channel,
00:05:54.11 we and others have been able to investigate the behavioral function
00:05:59.02 of the main olfactory system.
00:06:00.23 So, this is just to show you the expression of this TRPC2 ion channel.
00:06:08.05 What you see on this part of the slide is a section
00:06:13.14 through this tubular structure that is formed by the vomeronasal organ
00:06:19.11 and what you can see here is the neural epithelium that borders the lumen
00:06:23.19 through which the pheromones are, rise in contact to neurons.
00:06:29.18 And in red is immunostaining with the TRPC2 channel
00:06:34.17 and you can see that the protein is highly expressed and very specifically expressed
00:06:42.03 along the sensory terminal of the VNO neurons,
00:06:46.01 here seen even better, on a dissociated neuronal preparation,
00:06:53.00 you can see the sensory dendrite here, where the sensory, the receptors and the channels are,
00:06:59.16 so, you know, these expression patterns really suggest an important role of the TRPC2 channel
00:07:07.05 in the sensory transduction in the vomeronasal organ.
00:07:12.01 And the idea is that the two families of epithelial?? pheromone receptors, V1Rs and V2Rs,
00:07:22.26 when binding to pheromonal signal, lead to a signal transduction cascade
00:07:30.16 and in turn, to the opening of the TRPC2 channels.
00:07:34.26 So, by the knockout of the TRPC2 channel, one can completely abolish the signal transduction
00:07:41.08 and lead to an animal without a functional vomeronasal organ.
00:07:44.17 So, this is what we did, and the first experiment that we performed
00:07:50.15 when we obtained a mutant animal is to indeed validate the claim that the TRPC2 channel
00:07:57.02 is essential for VNO signal transduction.
00:07:59.10 And the experiment, so here is just the demonstration that in the TRPC2 mutant
00:08:06.11 there is no TRPC2 protein made anymore, compared to ubiquitous proteins, such as beta tubulin,
00:08:14.14 and in collaboration with Markus Meister, an actual physiologist in my department,
00:08:22.12 and Tim Holy, who was a post-doc in Marcus's lab,
00:08:26.22 we performed the electrical recording of the VNO neurons
00:08:30.29 in response to pheromonal stimuli.
00:08:34.16 So, the idea is to use a flat electrode array
00:08:41.07 in which each of the dots here represent a different electrode that can record from neurons
00:08:46.00 the electrical activity of neurons in the vicinity
00:08:48.27 and a VNO epithelium is pressed flat against this electrode array
00:08:54.20 and maintained by a mesh and then we can puff pheromonal stimuli
00:08:59.12 and record the activity of the neurons that have been stimulated by specific chemical cues.
00:09:05.28 And when we did the experiment and compared the situation
00:09:10.14 in the wild-type animal or the heterozygous animal to the situation in the mutant,
00:09:15.19 it became very clear that pheromonal stimuli leads to an increase in the spiking rate
00:09:21.24 of VNO neurons, are recorded by the electrode array,
00:09:25.17 but there was absolutely no stimulation in the TRPC2 mutant.
00:09:29.15 So, in other words, the VNO neurons are unable to respond to pheromonal stimuli.
00:09:34.27 We know that there are neurons here because if we stimulate the preparation with potassium chloride,
00:09:41.25 high concentration of potassium chloride,
00:09:43.15 we can see a very strong, non-specific neuronal firing that comes just from the depolarization of the cells
00:09:51.17 however, these cells are unable to specifically respond to pheromonal stimuli.
00:09:56.04 So, the VNO is basically silent and what we have is a mouse line
00:10:02.19 in which the olfactory detection can occur, but the vomeronasal detection is completely impaired.
00:10:11.22 So, what's happening to the behavior of these animals?
00:10:15.09 Well, to our big disappointment at first, this animal didn't seem to show any phenotype.
00:10:21.10 We expected, from surgical experiment, that animals without VNO
00:10:27.01 would be unable to mate. But, when we put a male mouse in the presence with a female,
00:10:33.02 a male mutant, in the presence of a female, they were mating perfectly normally,
00:10:37.11 in fact, exactly with the same frequency as wild-type animals.
00:10:43.03 So, we were very disappointed and even questioned what really,
00:10:46.26 what the vomeronasal organ good for?
00:10:49.29 And then we thought a little bit further and decided to study another set of behavior,
00:10:57.23 and we used very well known observation from Konrad Lorenz
00:11:03.27 that described behavior along these words.
00:11:09.25 If you put together, into the same container,
00:11:12.03 two sticklebacks, lizards, robins, rats, monkeys or boys,
00:11:15.26 who have not had any previous experience with each other, they will fight.
00:11:20.01 You can add to these two politicians, two scientists, two whatever,
00:11:25.11 when you put two males of any animal species in the same cage or room,
00:11:31.03 they will tend to fight with each other.
00:11:33.16 Well, we did this experiment
00:11:35.05 and together with the wild-type male mouse
00:11:42.07 and mutant male mice, and I'm going to show to you
00:11:44.20 the behavior or the mutant compared to that of the wild-type.
00:11:49.28 The behavioral paradigm that we use is as follows.
00:11:52.20 We know that in rodents, fighting behavior between two males
00:11:57.04 arises from the detection of male pheromones.
00:12:01.00 So, in order to set up an experimental system in which we can control the presence
00:12:10.29 or not of the male pheromones that trigger the male behavior,
00:12:16.29 our paradigm was chosen as follows.
00:12:20.10 We had a resident male, so an animal that stays in its cage for a couple of weeks,
00:12:27.28 and kind of established its territory, and we then introduced into that resident cage
00:12:35.09 an intruder. And the intruder is of different kind.
00:12:39.23 We first introduced a male intruder that is a castrated male.
00:12:44.29 Pheromones are under the control, pheromone production is under the control of testosterone,
00:12:50.01 and therefore, the castrated male is unable to produce any male pheromones.
00:12:55.19 And when we do this experiment, you can see in this video,
00:13:01.11 so you have a resident, the resident male,
00:13:06.21 and the castrated animal further here, the intruder does not emit any male pheromones,
00:13:14.19 and as you can see, these two mice just coexist very peacefully.
00:13:20.08 So, there's not even really seem to be any specific behavior of one animal versus the other.
00:13:30.04 Now, in the next video what you are going to see are the same two animals,
00:13:34.19 but the experimentator has put now 10 microliter of male pheromones
00:13:42.12 on the fur of the castrated intruder.
00:13:45.25 So, this castrated intruder does not naturally emit any pheromones,
00:13:51.10 but the pheromones, the male pheromones, is added exogenously,
00:13:55.04 and when this is done, you have now, the intruder here,
00:13:59.08 as you can see the resident detects the male pheromones
00:14:02.23 and immediately starts to fight.
00:14:05.29 So, the resident has adopted this defensive posture,
00:14:12.27 really doesn't understand what's happening to him,
00:14:15.15 and, as you can see, the resident is really a very aggressive,
00:14:21.14 and this is an extremely robust behavioral reaction,
00:14:25.12 which is a male detecting another animal emitting male pheromones will very brutally attack that animal.
00:14:32.15 Ok, so in the next, so that was the positive control,
00:14:37.09 this is what wild-type mice do, a male mouse detecting another male using olfactory cues
00:14:43.29 will attack that other males.
00:14:45.25 Now, if we use a TRPC2 male,
00:14:49.27 and so this is the male animal,
00:14:52.14 and this is the same intruder here that has been swabbed with urine,
00:14:56.29 what you see if strikingly different.
00:14:59.09 And I hope that even people without any experience in male behavior,
00:15:04.26 in mouse behavior, can very well visualize that what we have here
00:15:09.12 is absolutely not a fighting behavior, but instead a very surprising mating attempt
00:15:17.07 of the male mutant versus the other male.
00:15:20.07 So, this was extremely puzzling, extremely surprising,
00:15:25.19 the male mutant, instead of attacking the other male,
00:15:28.22 is trying to mate with it.
00:15:30.10 So, what's going on? Well, the key experiment was to actually put both male and female in the same cage.
00:15:37.29 So, again, if you have a male mutant in the presence of a female,
00:15:42.00 the mutant will mate perfectly normally,
00:15:44.18 but if you now put also a male in the cage, a male intruder,
00:15:50.22 what we discovered to our big surprise is that the mutant is unable to discriminate
00:15:56.22 between males and females and in fact, attempts to mate with each of them
00:16:02.08 with equal frequency.
00:16:03.19 And that led us to suggest, to propose, that the role of the vomeronasal organ
00:16:10.04 is not to trigger mating behavior, as was what was expected from the literature,
00:16:15.13 an animal without a functional VNO seemed clearly able to mate normally with a female,
00:16:21.11 but these animals seemed completely unable to discriminate between males and females.
00:16:27.26 And so, we even control for this behavior in a large arena
00:16:35.26 that you can see here. So, this is result from the observation that social behavior in general
00:16:43.25 can be very different in small cages or in more natural conditions.
00:16:48.11 So, we put a bunch of male mutants in the cage, and left them for several weeks,
00:16:53.14 just, you know, letting them do whatever they wanted,
00:16:56.18 for extended period of time and recording them constantly.
00:17:00.20 And as you can see, when we play the video,
00:17:03.19 is these males that form these courtship chains that are quite striking
00:17:08.22 in which one male is trying to copulate with the male in front,
00:17:13.10 and is trying to be copulated with the male just behind.
00:17:16.28 Those are extremely striking behavior that can go on for several minutes.
00:17:22.17 Now, you know, I'm showing this for entertainment value, but also for a very interesting purpose also,
00:17:31.29 which is that these courtship chains that are observed in the mouse
00:17:35.22 are actually strikingly similar to the courtship chains that have been observed in Drosophila
00:17:42.27 in a particular mutant called the fruitless mutant.
00:17:46.01 So, fruitless is a transcription factor that has many splicing variants,
00:17:50.05 some of them are sexually dimorphic, and the mutation of the male specific splicing variants
00:17:59.00 lead to these male flies that show these male-male courtship chains
00:18:05.03 that are indeed very similar to what we've observed in the TRPC2 mutant mice.
00:18:13.02 Now, this is very striking because fruitless is a transcription factor
00:18:16.17 that is expressed very widely within the brain
00:18:19.24 and is thought to be responsible for the development of the neuronal circuit
00:18:28.03 that enables courtship behavior, and the TRPC2 channel is an ion channel
00:18:33.02 expressed only in sensory neurons that give information about the gender of the animals.
00:18:38.24 And I really very clearly found the similarity of the behavior very striking.
00:18:45.08 Obviously it's a very interesting question of whether or not the mammalian brain
00:18:50.24 is expressing a fruitless equivalent,
00:18:53.27 and so far, nobody has really been able to find interesting candidates.
00:18:59.08 So, from this study, we propose a model of the control of mating behavior,
00:19:09.11 reproductive behavior, in the mouse that is quite different from the classical view
00:19:14.08 of the role of the vomeronasal system. What we found is that sensory cues
00:19:20.21 that are independent from the vomeronasal system are sufficient to trigger mating behavior.
00:19:27.08 And the role of the vomeronasal organ is to provide another type of information
00:19:34.07 which is gender identification.
00:19:35.27 So, here, there are really two systems that work with each other.
00:19:40.08 One is the vomeronasal information that provides information about gender,
00:19:45.11 and the other one, some other sensory signal that is sufficient to trigger mating behavior.
00:19:50.25 And very clearly, in the absence of vomeronasal information,
00:19:55.20 the default behavior is mating behavior.
00:19:58.12 And when a male is encountering another male,
00:20:02.09 then the vomeronasal cues detect signals that say no mating behavior,
00:20:08.07 but instead aggressive behavior.
00:20:10.11 And so, this, obviously is quite different from the classical view of the vomeronasal organ
00:20:19.01 in triggering mating behavior and I will come back later during the talk
00:20:24.24 on some possible explanation of the discrepancy between the results obtained with TRPC2 channel,
00:20:31.02 so, genetic ablation,
00:20:33.08 compared to what has been obtained with classical surgical ablation.
00:20:37.17 So, I'll tell you a little bit why I think those results were different
00:20:43.12 and this comes from results that we obtained very recently
00:20:46.23 and that I will describe in the third part of the talk.
00:20:49.06 Now, this is obviously very striking,
00:20:53.07 this shared work between vomeronasal system and other sensory cues
00:20:59.27 in the control of gender identification and at that point,
00:21:05.02 I really was curious, how are other animals distinguishing the sex of their conspecifics?
00:21:12.21 So, I went into the literature and investigated a little bit what people had described
00:21:17.09 in other species. And here is what I found.
00:21:19.27 So, in this particular species of bird, the shell parakeet,
00:21:24.20 this is a female, and this is a male.
00:21:30.07 And the animal, the parakeet, recognize the gender of their conspecific
00:21:35.20 based on the presence of these blue dots on the top of the beak.
00:21:40.14 And this particular blue dot is essential for sex identification,
00:21:46.08 which is that if you paint a blue dot on the beak of a female,
00:21:54.16 the animal now is identified as a male, and other males will attack that female with a blue dot,
00:22:02.00 thinking it is a male.
00:22:03.21 And similarly, if you mask the blue dot from the beak of a male,
00:22:09.23 then the other males are going to try to mate with this male without the blue dot,
00:22:14.27 by thinking it's a female.
00:22:17.01 So, the blue dot, the visual cues, the identification of this blue dot,
00:22:21.05 is essential to the identification of this animal as a male or a female.
00:22:25.23 Similarly, in this other species of bird, the American flicker,
00:22:31.12 here's a female, here's a male,
00:22:33.18 and what provides the identification of one being male, the other one a female,
00:22:38.16 is the presence of the black moustache.
00:22:40.21 So, if you were to mask the black moustache on the male,
00:22:44.26 this animal would be identified as a female
00:22:47.19 and the other males will try to, attempt to, copulate with this male without a moustache
00:22:53.14 and this animal here that is a female, if you plant a black moustache,
00:22:58.15 males will attack that female, thinking it's a male.
00:23:01.27 And Tinbergen, who was a very famous etiologist,
00:23:05.23 described this by calling those signs the badges of masculinity,
00:23:11.09 which is here, this is my blue dot, or this is my moustache, I'm a male, and if I don't, then I'm a female.
00:23:17.10 And so this is quite interesting, the visual recognition of the gender identity,
00:23:24.23 and I think that what we found for the pheromones is the olfactory equivalent
00:23:31.19 of the badge of masculinity, which is the vomeronasal organ is responsible for
00:23:36.16 discriminating between males and females.
00:23:39.17 Now, obviously the animal we care the most about are humans.
00:23:44.25 Do human, what kind of strategy do humans use
00:23:48.02 and is the vomeronasal organ also being used for sex identification?
00:23:54.13 And here, things are likely to function very differently.
00:23:58.18 The TRPC2 channel which is responsible for the function of the vomeronasal organ
00:24:04.09 in rodents is a non-functional gene in humans
00:24:08.12 and actually in higher primates.
00:24:10.17 This little triangle that you see here, there are 9 of them,
00:24:15.05 are the site of deleterious mutations such that higher primates and humans
00:24:22.05 have a number deletion or frame shift or nonsense mutation
00:24:27.24 that make this gene unable to generate a functional protein.
00:24:33.27 And it's quite interesting to actually look throughout evolution,
00:24:38.18 when these mutations occurred. And this is this work of the laboratory of Emily Limon
00:24:45.28 that shows that the mutation really starts to accumulate
00:24:50.19 at the split between new world monkeys and old world monkeys and apes,
00:24:55.27 so all these part of the tree of higher primates really is unlikely to use the vomeronasal organ
00:25:04.13 as a tool to discriminate sex.
00:25:09.04 And I think what's quite interesting and was proposed by Emily Limon's group
00:25:15.07 is that this split here between the new world and the old world monkeys
00:25:22.12 also correspond to the duplication of the red and green opsin genes
00:25:28.14 such that animals in this part of the tree here have an additional opsin receptor gene
00:25:35.13 and therefore, the ability now to discriminate between two colors,
00:25:40.13 red and green, whereas animals in this part of the tree have one opsin gene
00:25:46.21 that detects both green and red color, and so this is being perceived as one particular color,
00:25:53.11 whereas here, those animals can discriminate between these two wavelengths of photons
00:25:59.29 that are very close to each other, but if distinguished by distinct receptors,
00:26:05.02 can now appear as distinct colors.
00:26:08.09 And this, from an evolutionary point of view
00:26:11.14 can provide an enormous advantage.
00:26:13.23 For example, the ability to discriminate between a ripe and a non-ripe fruit,
00:26:19.27 ripe fruit is full of calories, full of sweet,
00:26:23.06 and this is obviously very advantageous for animals that are able to distinguish those highly nutritious food
00:26:31.20 from a non-ripe fruit that doesn't have all this properties.
00:26:39.02 So, to come back to our two systems, we've seen using genetic mutation
00:26:47.07 that the vomeronasal system is specialized into detecting the sex identity of individuals,
00:26:55.28 individual animals, and that the dichotomy
00:27:02.16 between pheromone perception in the olfactory, in the vomeronasal system,
00:27:07.20 is not that absolute in other words,
00:27:11.10 that mating behavior can occur without the vomeronasal organ,
00:27:14.21 therefore, there has to be something else that provides information to the animal
00:27:19.21 about the presence of conspecific and the ability to mate.
00:27:24.02 Now, how are we going to go explore the system further?
00:27:29.11 Well, as I mentioned, we and others have identified a specific receptor
00:27:35.11 for chemicals detected in the vomeronasal organ and Richard Axel and Linda Berg
00:27:41.08 discovered the olfactory receptors responsible for detection in the main olfactory system
00:27:46.17 and so, one really interesting goal is to try to understand what are the chemicals
00:27:54.03 that are detected and provide animal with information about the gender identity of the animal
00:27:59.20 or which one provides information that leads to aggressive behavior,
00:28:07.00 or any type of behavior that is triggered by these two systems.
00:28:13.11 And this direct question, what ligand generate what type of behavior, is a little bit difficult to address right now
00:28:25.25 because of technical difficulty into expressing pheromone receptors in vitro
00:28:32.05 and therefore, finding an easy, high-throughput assay to identify the ligand of these receptors.
00:28:38.26 And so, what the strategy that my lab decided to use
00:28:45.25 is start in the center of the brain, instead of the peripheral organ.
00:28:51.08 So, instead of trying to identify what are the receptors involved in specific behaviors,
00:28:55.21 and then trying to go and follow the circuitry in the brain,
00:28:59.12 we decided to do exactly the opposite, which is it well known that specific nuclei in the hypothalamus
00:29:06.14 are involved in aggressive behavior or reproductive behavior
00:29:10.27 and we therefore decided to trace the input to those specific centers,
00:29:17.23 in other words, what are the areas of the brain
00:29:21.04 and what are the specific neurons of the vomeronasal organ or olfactory epithelium
00:29:25.28 that send input, processed by different brain circuitry, that end up in, let's say,
00:29:32.08 an area of the brain involved in reproductive behavior or in aggressive behavior.
00:29:40.00 This requires two parameters.
00:29:44.01 One is the type of neurons in the brain that we want to investigate,
00:29:50.00 so, a specific set of neurons that are clearly involved in either reproduction
00:29:55.18 or the control of aggressive behavior,
00:29:58.25 and the second parameter is to find a tool that enable to link this particular set of neurons
00:30:06.10 to the connected circuit in the brain.
00:30:09.29 So, the set of neurons that we decided to study first
00:30:14.09 are neurons that express, release, express and release
00:30:19.19 a very particular neuropeptide called luteinizing hormone releasing hormone.
00:30:25.18 This is a neuropeptide that is expressed by a very small population of neurons
00:30:31.12 in the medial preoptic area in the hypothalamus.
00:30:33.20 They might be as few as six or seven hundred of these neurons
00:30:39.15 that are dispersed within this very large area of the hypothalamus,
00:30:43.15 called the medial preoptic area.
00:30:45.08 These neurons synthesize LHRH and release it in the portal vein
00:30:53.28 and the neuropeptide LHRH is absolutely essential for the control of fertility and reproduction
00:31:01.13 in vertebrates.
00:31:03.18 So, animals that are deficient in LHRH are sterile and do not develop their gonads,
00:31:12.18 functional gonads, and are impaired in sexual behavior.
00:31:15.23 The function of these cells is as follows.
00:31:18.25 They release LHRH into the portal vein that will then interact with neurons with cells in the pituitary gland
00:31:29.07 and lead to the release of LH and FSH that is in turn is released into the blood
00:31:35.17 and lead to the development of the function of the gonad,
00:31:39.29 both males and female gonads.
00:31:42.00 These functional gonads will in turn release steroid hormones,
00:31:46.19 the steroid hormones will, on one hand, lead to the development of the secondary sexual traits
00:31:53.09 and also provide feedback to the brain in both enhancing sexual behavior
00:32:00.14 but also providing a feedback to the release of LHRH.
00:32:04.20 LHRH also directly communicates with other areas of the brain
00:32:09.19 by synaptic contact and are essential for sexual receptivity
00:32:13.22 and modulate sexual behavior.
00:32:20.00 Now, what is quite interesting is that these neurons are really the master regulators
00:32:24.26 of reproduction and fertility in the animals
00:32:27.25 and therefore, their own function is very tightly regulated.
00:32:32.15 They are sensitive to both the internal and external state of the animal.
00:32:37.22 So, one interesting factor that controls their function
00:32:44.22 is actually a very unknown set of factors that is a developmental clock
00:32:50.28 that triggers puberty. So, these neurons are not functional before puberty
00:32:56.13 and then at some point, they become functional and release LHRH at high frequency.
00:33:04.05 The developmental clock that triggers this function is not well understood at all
00:33:10.12 but, for sure, this is one of the main controls of the function of LHRH neurons.
00:33:16.03 Now, these neurons are also sensitive to external cues,
00:33:21.06 for example, pheromonal cues or sensory stimuli that then leads to reproductive behavior.
00:33:28.17 So, it is now that pheromone detection leads to increased LHRH synthesis
00:33:34.20 and increased LHRH release.
00:33:38.08 Now, moreover, the function of these neurons is also extremely sensitive
00:33:43.29 to the internal state of the animal.
00:33:47.22 For example, an animal that is starving is not going to reproduce,
00:33:52.03 or an animal that is very stressed won't reproduce either
00:33:55.22 and this is because the other areas of the brain that deal with stress
00:34:00.25 or the level of nutrition are sending signals
00:34:03.28 that tightly control these LHRH neurons.
00:34:08.06 So, you know, the hypothalamus in general is in charge of the homeostasis of the animal
00:34:13.05 and the appropriate coordination of all the functions of the organism,
00:34:18.27 reproduction, aggression, nutrition, sleep, et cetera.
00:34:22.28 And so it's very essential, the release of LHRH has many levels of control,
00:34:28.27 both by the environment and the internal state of the animal.
00:34:34.06 So, we decided that these neurons would be a perfect target for a study
00:34:41.17 and that by investigating all the, the nature of the sensory cues that send input to those.
00:34:49.17 we will understand better the circuitry controlling reproduction and fertility in the rodent brain.
00:34:56.10 Now the second set of tools that we used are viruses
00:35:02.02 and in particular a set of viruses called pseudo rabies viruses that have the ability to replicate within neurons
00:35:09.19 and more importantly to jump across synaptically connected chains of neurons.
00:35:18.16 So, it will replicate into a neuron, then cross the synapse and reach from post-synaptic to pre-synaptic cells
00:35:28.00 and then jump again, et cetera,
00:35:30.02 and will therefore infect all the neurons that are connected to each other synaptically.
00:35:34.26 Now, the type of virus that we used
00:35:38.20 are conditional pseudo rabies viruses that have been made by Lynn Enquist
00:35:42.19 in Princeton and also Jeff Friedman at Rockefeller,
00:35:48.21 they built modified pseudo rabies virus
00:35:53.25 that when infecting the neuron, is non-functional. So, that virus, shown here in red,
00:36:02.00 infect the neuron, sorry, but is unable to replicate,
00:36:07.02 and when a neuron expresses a particular enzyme called a Cre recombinase,
00:36:12.20 that is indicated here like a pair of scissors,
00:36:15.26 it cut out a cassette of the virus genome, that makes now the virus able to replicate
00:36:25.04 and also to express the green fluorescent protein
00:36:28.15 and therefore now, the virus can replicate, jump across synapses,
00:36:32.22 and label all the infected neurons in green, fluorescent green.
00:36:37.00 And therefore, we generated a transgenic mouse line
00:36:42.13 expressing the Cre recombinase specifically in neurons expressing LHRH.
00:36:49.11 So, LHRH promoter driving the Cre recombinase
00:36:52.14 and by injecting this conditional virus in the medial pre-optic area,
00:37:00.06 the virus will infect neurons, but replicate and become green fluorescent
00:37:04.15 only in neurons expressing the neuropeptide LHRH
00:37:09.02 and therefore, one will be able to visualize very nicely all the LHRH neurons,
00:37:16.02 but also all the afferent neurons, the neurons synaptically connected to this LHRH neurons.
00:37:23.08 And so, when we performed the study, we identify a lot of infected neurons throughout the brain,
00:37:33.02 indicating that they were synaptically connected,
00:37:36.17 sending information into LHRH neurons.
00:37:40.05 And as control, we could visualize a number of brain areas
00:37:47.08 that were known to send information to LHRH neurons,
00:37:50.09 in particular, areas that provide sensory input,
00:37:55.26 that provide information about the circadian clock, suprachiasmatic nucleus
00:38:01.08 that provide information about the stress level in the brainstem
00:38:06.04 or the level of nutrition in the, from the arcuate nucleus and other areas.
00:38:13.12 So, we found all of these by infecting the, injecting the virus
00:38:18.25 by LHRH neurons, we were able to identify all these brain areas,
00:38:24.01 but our main goal was to try to investigate within the vomeronasal system
00:38:31.25 all the specific brain areas that ultimately send input to the medial preoptic area,
00:38:40.09 the neurons expressing LHRH neurons there.
00:38:42.17 And specifically, what we were hoping is that if the virus could indeed
00:38:47.20 jump from post-synaptic to presynaptic cells,
00:38:51.04 throughout enough jumping of synapses,
00:38:54.22 we could even be able to recognize specific populations of the vomeronasal organ
00:38:59.25 and maybe recognize them with the green fluorescent protein,
00:39:03.16 maybe identify what are the specific receptors that ultimately send information to LHRH neurons.
00:39:09.11 And when we performed the experiment,
00:39:11.28 when I say we, actually my graduate student Hayan Yoon,
00:39:16.12 the result was quite astonishing. Which is that we recognized many brain areas,
00:39:22.18 but within overall the olfactory and vomeronasal system,
00:39:27.19 the brain areas that we recognized in the cortical amygdala, the pyriform cortex,
00:39:34.00 the olfactory tubercule, the olfactory bulb,
00:39:36.09 and a specific population, also, very smallest population of neurons in the olfactory neurons.
00:39:41.08 Now, what's wrong here?
00:39:43.13 Well, what's wrong is that all of these belong to the main olfactory system,
00:39:48.07 and we were actually completely unable to recognize, to identify any labeled area
00:39:55.14 within the vomeronasal system. Which is that in contrast to what was found in the literature,
00:40:03.05 in which classical dye tracing experiment had identified strong connection of the medial pre-optic area
00:40:11.12 to the medial amygdala and the vomeronasal system,
00:40:15.08 what we found was instead very strong connection to the main olfactory system
00:40:20.24 and in particular, to cortical areas of the olfactory system.
00:40:26.00 So, so what's going on? Well, you know, classical dye tracing experiments
00:40:31.16 put the dye in the particular area where the LHRH neurons are located,
00:40:36.19 them among many other types of neurons,
00:40:40.01 and these tracing experiment indeed show a very strong link
00:40:44.02 between the vomeronasal system and the medial preoptic area
00:40:47.22 in which LHRH neurons are located.
00:40:49.25 Our experiment is a genetically controlled tracing experiment
00:40:53.26 in which what we visualize are the very specific connections
00:40:57.14 of these very precise population of neurons expressing the LHRH genes.
00:41:03.18 And when we do this, what we found is that it's possible that all the other neurons around
00:41:08.13 are connected to the vomeronasal system,
00:41:10.03 but the LHRH neurons are precisely not connected to the vomeronasal system
00:41:16.04 and instead are connected to the main olfactory system.
00:41:19.26 So, this is quite interesting and in particular,
00:41:26.27 it points to a particular population of neurons in the olfactory system
00:41:33.24 that send input to LHRH neurons, to neurons involved in the control of reproduction,
00:41:43.13 and therefore, these neurons are very likely to detect pheromones.
00:41:47.21 In fact, by definition,
00:41:49.04 if they are connected to areas of the brain involved in the control of reproduction,
00:41:54.19 they are pheromone detecting neurons.
00:41:57.02 We've recognize that these neurons expressing LHRH,
00:42:01.28 which is essential for the control of reproduction,
00:42:04.12 are, have this massive connection from the olfactory system.
00:42:08.15 This is obviously an anatomical finding and it's absolutely essential to have some type of functional correlates.
00:42:18.15 In other words, if indeed the main olfactory system provides such a massive input
00:42:24.11 to LHRH neurons in the medial preoptic area,
00:42:27.06 then animals that are deficient in olfactory function should also have deficiency in reproduction.
00:42:35.12 And this is indeed exactly what we found.
00:42:37.18 Remember, we found that the TRPC2 mutant male are perfectly able to mate,
00:42:44.12 but mate both with males and females.
00:42:47.04 And when we now investigated male mouse
00:42:50.28 that are deficient for the olfactory cyclic nucleotide gated channel,
00:42:55.05 so impaired in the main sense of smell,
00:42:58.09 what we found was that these animals are absolutely unable to mate.
00:43:03.19 So, they don't even recognize the female, don't even acknowledge the presence of females.
00:43:07.29 So, these provide very nice and direct functional correlate
00:43:14.11 to this massive input for the main olfactory system
00:43:18.08 into the control of reproduction.
00:43:20.14 So, in other words, it's now very clear that both vomeronasal system
00:43:26.14 and olfactory system both contribute to the perception of pheromones
00:43:31.07 that lead then to the control of reproduction and fertility in the animal.


Sufficient evidence, much still accumulating, suggests the presence of four types of pheromones in human chemical communication. These include primers, signalers, modulators, and releasers. Initially, we discuss potential sources of these cues in humans. We then explore the notion that detection of pheromones among humans is via the vomeronasal organ (VNO an unlikely possibility) and close with a discussion of human responses to pheromones.

Source and Signal: Axillary Chemistry and Pheromone Creation

The axilla is a unique source of human odor. In addition to a high density of eccrine glands, the axilla contains large numbers of sebaceous and apocrine glands (Labows et al., 1982 ). The interactions between the cutaneous microflora and skin secretions lead to a complex mix of odorants (Leyden et al., 1981 Labows et al., 1982 ).

As seen in Figure 1, human axillary extracts contain a complex mixture of volatile chemicals. One or more of these volatile molecules may have pheromonal function. Axillary secretions and odorants appear to be ideal sources of pheromones: they are secreted to an area that often contains hair that can greatly increase the surface area for dispersal, are warmed to aid in volatilization, and are positioned nearly at the level of the nose of the recipient when near another person. The axilla is also the focal point for a multibillion-dollar consumer product industry. These factors, both fundamental and applied, have motivated research aimed at identifying the nature, abundance, and biogenesis of the odorous and nonvolatile components found in the underarm.

A gas chromatographic trace of an extract of male axillary secretions. Much is known about axillary chemistry. For example, many of the peaks in the chromatogram have been chemically identified. Furthermore, for many of the compounds, the origins and hence the formation of odor are also understood. Specifying pheromonal components within the GC trace, if they are even visible therein, awaits identification via bioassay-driven methodology. To this end, chemistry alone may not be sufficient. For example, results from bioassays may suggest active components where no peaks appear on the chromatogram, which has been known to occur when trying to identify the active components in foods or fragrances from flowers. In the end, use of the human nose coupled with a biological response and chemical analyses should prove to be successful.

More than a decade of research has presented both organoleptic and analytical evidence that a mixture of C6–C11 normal, branched, and unsaturated acids present in axillary sweat constitutes the characteristic axillary odor. The details of the chemical identification, exact structures, and synthesis (of noncommercially available compounds) have been described (Zeng et al., 1991 , 1992 ). In terms of relative abundance, these acids, in particular (E)-3-methyl-2-hexenoic (E-3M2H), are present in far greater quantity than volatile steroids, e.g., androstenone, which were previously thought to be important axillary odors (Gower and Ruparelia, 1993 ). In samples of secretions from the axillae of males that were combined before analysis, the concentration of E-3M2H was approximately 357 ng/μl extract, whereas that of androstenone was 0.5 ng/μl extract (Zeng et al., 1996b ). In combined samples from females, the straight-chain acids were present in greater relative abundance than E-3M2H. Further, no androstenone was detected in these extracts. A related steroid, androstenol, was present (3.5 ng/μl extract), albeit in far lower concentration than E-3M2H (150 ng/μl extract) or the other acids (Zeng et al., 1996b ). The Z-isomer of 3M2H was also present in the extracts from each gender, but in different relative abundance: 10:1 (E:Z) in males and 16:1 (E:Z) in females. E-3M2H and androstenone have comparable low olfactory thresholds (Baydar et al., 1992 Gower and Ruparelia, 1993 Wysocki et al., 1993 Zeng et al., 1996b ).

Recently, researchers at Givaudan (Natsch et al., 2003 ) identified 3-methyl-3-hydroxylhexanoic acid (HMHA) as an additional important axillary odor constituent however, the olfactory threshold for this compound has not yet been reported. Qualitatively, this compound has a cumin-like, “sweaty” note reminiscent of E-3M2H, but more pungent (data not shown).

The precursors to axillary odor reside in the apocrine glands (Labows et al., 1982 Zeng et al., 1992 , 1996a , 1996b ). The characteristic axillary odor is formed from the interaction of odorless (water-soluble) precursor molecules found in apocrine secretion with the cutaneous axillary microorganisms (Labows et al., 1982 Zeng et al., 1992 ). In addition, it has been demonstrated that the 3M2H is carried to the skin surface bound to two proteins that have been designated apocrine secretion odor-binding proteins: ASOB1, apparent molecular weight 45 kDa, and ASOB2, apparent molecular weight 26 kDa (Spielman et al., 1995 , 1998 ). The polypeptide chain of ASOB2 is identical to apolipoprotein D (ApoD), a known member of the lipocalin proteins. The ligand carried by the apocrine ApoD is 3M2H. The structure of ASOB1 remains to be fully elucidated but it too appears to carry acidic molecules. The Givaudan group (Natsch et al., 2003 ) has suggested that a nonodorous precursor they isolated, an amide of 3-methyl-3-hydroxylhexanoic acid and glutamine (Nα-3-hydroxy-3-methyl-hexenoyl-glutamine HMHA-Gln), is the actual precursor. However, due to their collection procedure, it is difficult to say that the 3M2H and/or HMHA is not initially intercathelated within ApoD.

The studies cited above, which detail the nature and origin of axillary odor, demonstrate the complexity of the components present in either axillary extracts or collected on T-shirts. They further demonstrate the similarity between human axillary secretions and nonhuman mammalian odor sources where lipocalins carry chemical signals used in pheromonal communication. In rodents (Novotny, 2003 ), pigs (Spinelli et al., 2002 ), and hamsters (Singer et al., 1989 ), volatile molecules appear to be bound to lipocalin proteins that transport them and are in part responsible for some of the activity. Hence, the chemistry of human axillary secretions appears to be analogous to other mammalian pheromone systems—an interesting and thought-provoking analogy. However, no bioassay-guided study has led to the isolation of true human pheromones, despite claims appearing in popular media (e.g., Web sites) and even suggested in some peer-reviewed articles (Sobel et al., 1999 Grosser et al., 2000 Savic et al., 2001 ). The axillary extracts discussed above may be thought of as a “medicinal tea” whose active ingredients remain to be isolated, much like the tea made from the extract of the foxglove plant that was given to chest-pain sufferers during the 18th and 19th centuries (Krantz, 1974 ). From this tea came the isolation and identification of digitalis.

The axillary constituents most often cited as putative human pheromones are volatile steroids: androstenone, androstenol, and 4,16-androstadien-3-one (androstadienone). The concentration and biogenesis of these compounds in human axillae have been examined (Rennie et al., 1991 Gower and Ruparelia, 1993 ). Additionally, androstenone and androstenol were found to be present in the characteristic odor fraction, at levels 50–100 times below the concentration of 3M2H and other organic acids (Zeng et al., 1992 ). Shinhoara et al. ( 2000 ) found that androstenol (commercially obtained) could alter LH pulsing when applied to the upper lip/nares region of female recipients at concentrations 1,000× above endogenous concentrations. Similarly, Jacob and McClintock ( 2000 ) used concentrations (of commercially available androstadienone) that were also 1,000× above reported axillary concentrations to demonstrate modulator pheromone effects for androstadienone. Subsequent work by Lundstrom et al. ( 2003b ) has demonstrated that the concentration used by Jacob and McClintock ( 2000 ) yielded vapor-phase concentrations of androstadienone that are about at the average olfactory threshold for this compound, namely, 211 vs. 250 μM used by Jacob and McClintock ( 2000 ). Lundstrom et al. ( 2003a ), however, did report a single significant mood effect (“being focused”) when they applied 250 μM to the nasal area of subjects.

Although the specific chemical identities remain to be determined, humans carry with them unique chemical signatures. These odorprints are hypothesized to consist of a bouquet of odorants whose relative amounts differ across individuals. These odorants may also be present in all of our bodily fluids and secretions and are regulated and/or produced in part by the set of genes that code for immune function (human leucocyte antigen HLA). Several studies have demonstrated that axillary volatiles collected on pads and/or T-shirts allow individuals to identify their own odor as well as those of their spouse and close kin (Schleidt, 1980 Porter and Moore, 1981 Schleidt et al., 1981 Cernoch and Porter, 1985 Hepper, 1988 ). These studies strongly suggest that axillary secretions contain odorants unique to individuals that may be used for identification (signaler pheromone). Some have suggested that they may play a role in mate choice (Jacob et al., 2002 ). HLA-related proteins have been detected in both the lactiferous ducts of the breast, a structure analogous to the axillary apocrine glands, and the intradermal portion of the sebaceous glands (Murphy et al., 1983 ). Studies from one laboratory (Zavazava et al., 1990 , 1994 ) have reported the presence of an HLA class 1 molecule in human axillary sweat collected after exercise (a mixture of apocrine, apoeccrine, sebaceous, and eccrine secretion). These investigators also demonstrated that individuals who were HLA-A23, -A24, or -B62 expressed higher levels of soluble HLA molecules in serum than individuals without those specificities. Two-thirds of individuals who had the strongest body odors, when evaluated organoleptically, were from one of the above antigenic specificities, suggesting a direct link between body odor intensity and levels of soluble HLA-related proteins. The only study that has examined the structures of immune system-related odorants was performed with rodents (Singer et al., 1997 ). Data in this publication suggest that in these animals the urinary odorprint is formed by acidic constituents. Phenylacetic acid was the sole identified acidic compound that was significantly different between the two groups with different MHCs. We currently hypothesize that human odorprints will also be formed by ratios of organic acids in the axillae, urine, and other fluids.

Pheromone Receptor: Likelihood of a Human VNO

Among mammals in general, the VNO is involved in the detection of pheromones (Table 1), and this is likely the case in at least some nonhuman primates (see Alport, 2004 in this issue for further discussion). However, the olfactory system also detects pheromones. In pigs, Dorries et al. ( 1997 ) reported that sows responded to the boar pheromone, androstenone, after reception by the VNO was prevented. In the lesser mouse lemur (Microcebus murinus), a prosimian that possesses a well-developed VNO, responses to chemical cues were mixed after disruption of inputs via the VNO, namely, female-elicited intermale aggression was eliminated, male investigation of females was reduced, and copulations with females was reduced. However, successful inseminations were not significantly different from control levels (Aujard, 1997 ). In mice, removal of the VNO (VNX) did not affect ability to learn a Y-maze-based task for a reward where reinforcement was provided upon successful chemosensory-based discrimination of MHC-type signals originating from urine obtained from donor mice mice with VNX continued to discriminate MHC-based individuality among other mice (Wysocki et al., 2004 ).

Pheromone responsea a References not intended to be exhaustive.
VNO involved Referencea a References not intended to be exhaustive.
Primer effects
Acceleration of puberty
Mouse Yes Lomas and Keverne ( 1982 )
Vole Yes Wysocki et al. ( 1991 )
Estrus synchrony Yes Sánchez-Criado ( 1982 )
Pregnancy failure Yes Brennan et al. ( 1990 )
Testosterone surge Yes Wysocki et al. ( 1983 )
Mating in sows No Dorries et al. ( 1997 )
Matting by male mice Yes Del Punta et al. ( 2002 )
Individual recognition No Johnston and Rasmussen ( 1984 )
Yes Steele and Keverne ( 1985 )
Recognition of MHC No Wysocki et al. ( 2004 )
Strain differences in Mice Yes Luo et al. ( 2003 )
Mood or emotion Not demonstrated

Among sheep, results of tests of VNO involvement in maternal behavior are mixed. Levy et al. ( 1995 ) generated a strong case for olfactory involvement. They had earlier reported that cutting the vomeronasal nerves had no effects on maternal behaviors. Notably, primiparous and multiparious ewes continued to discriminate own from alien young, whereas rendering olfaction nonfunctional significantly disrupted maternal behaviors. Booth and Katz ( 2000 ) later reevaluated a role for the VNO in similar situations by cauterizing the opening of the VNO, thereby preventing access of chemosensory stimuli to receptor cells therein. As stated by the authors: “Cauterized ewes allowed alien lambs to suckle and they were unable to distinguish alien lambs from their own lambs, whereas the ewes … with functional vomeronasal organs … violently rejected any alien lamb's attempt to suckle. Thus, female sheep use their vomeronasal organs for neonatal offspring recognition” (Booth and Katz, 2000 : p. 953).

Importantly, the VNO also detects nonpheromonal chemicals (Tucker, 1971 Sam et al., 2001 ). Therefore, linking detection of pheromones with the VNO or labeling substances detected by the VNO as pheromones is a non sequitur (Preti and Wysocki, 1999 Wysocki and Preti, 2000 , 2002 ).

Some have claimed that the human VNO is the detector of human pheromones (Monti Bloch and Grosser, 1991 Monti Bloch et al., 1994 Berliner et al., 1996 ). Supporting evidence comes from electrophysiological recordings obtained from the epithelium within the adult VNO (Meredith, 2001 ). These findings are puzzling, given the overwhelming preponderance of genomic, proteomic, and anatomical evidence strongly suggesting that the human VNO is nonfunctional, at least in the way that it is understood to work from studies in other mammals (Table 2).

Level Nonhuman Humana a In some instances, references are only a sampling of what is available.
Fetus Adult
Vomeronasal organ (VNO) Tubular structure in rostral nasal cavity Presentb b Boehm and Gasser ( 1993 ) Boehm et al. ( 1994 ) Smith et al. ( 1996 , 1997 ).
Presentc c Jacobson ( 1811 ) Takami et al. ( 1993 ) Smith et al. ( 1998 ) Bhatnagar et al. ( 2002 ) Smith et al. ( 2002 ).
Bipolar receptor cells within VNO Typically bilateral on medial surface Presentd d Kjaer and Fischer Hansen ( 1996a , 1996b ).
Absente e Trotier et al. ( 2000 ) Witt et al. ( 2002 ).
Intact receptor genes presumed to be expressed in VNO At least two subfamilies, namely, V1R and V2R (≈ 150 in V1R alone) Unknown Absentf f One V1R1L gene is expressed in the olfactory epithelium (Rodriguez et al., 2000 ) others may be intact, but expression has not been identified in the VNO (Rodriguez and Mombaerts, 2002 ).
Transduction mechanisms Uses TRP2 Ca ++ channel Unknown Absentg g Liman and Innan ( 2003 ).
Axonal projections to brain (from bipolar neurons) Traverse nasal septum and cross cribriform plate rostromedially Presenth h Kjaer and Fischer Hansen ( 1996a , 1996b ).
Absenti i Inferred from Boehm et al. ( 1994 ), who note that the vomeronasal nerve disappears during development of the fetus, after neurons that contain GnRH complete their migration from the VNO to the olfactory bulbs and basal forebrain (Schwanzel-Fukuda, 1999 Wray, 2002 ).
Identifiable accessory olfactory bulb Typically in rostrocaudal location in olfactory bulb Unknown Absentj j Meisami and Bhatnagar ( 1998 ).
  • a In some instances, references are only a sampling of what is available.
  • b Boehm and Gasser ( 1993 ) Boehm et al. ( 1994 ) Smith et al. ( 1996 , 1997 ).
  • c Jacobson ( 1811 ) Takami et al. ( 1993 ) Smith et al. ( 1998 ) Bhatnagar et al. ( 2002 ) Smith et al. ( 2002 ).
  • d Kjaer and Fischer Hansen ( 1996a , 1996b ).
  • e Trotier et al. ( 2000 ) Witt et al. ( 2002 ).
  • f One V1R1L gene is expressed in the olfactory epithelium (Rodriguez et al., 2000 ) others may be intact, but expression has not been identified in the VNO (Rodriguez and Mombaerts, 2002 ).
  • g Liman and Innan ( 2003 ).
  • h Kjaer and Fischer Hansen ( 1996a , 1996b ).
  • i Inferred from Boehm et al. ( 1994 ), who note that the vomeronasal nerve disappears during development of the fetus, after neurons that contain GnRH complete their migration from the VNO to the olfactory bulbs and basal forebrain (Schwanzel-Fukuda, 1999 Wray, 2002 ).
  • j Meisami and Bhatnagar ( 1998 ).

Most of the genes identified as coding for receptor proteins in the VNO of the mouse are pseudogenes in humans (Rodriguez and Mombaerts, 2002 ). Furthermore, although a few genes that express receptors in the mouse VNO appear to have an intact coding region in the human genome (Rodriguez et al., 2000 ), none have been found to express proteins within the human VNO.

Among mammals that express functional receptors within the membranes of bipolar receptor cells of the VNO (Fig. 2), sensory transduction associated with these molecular receptors appears to rely on a calcium channel that is encoded by the trP2 gene (Liman and Innan, 2003 ). Among humans and other catarrhines, trP2 is a pseudogene (Liman and Innan, 2003 ). Hence, at the genomic and proteomic levels, the human vomeronasal system cannot function as it is understood to work in nonprimates.

Coronal sections through the VNO, courtesy of T.D. Smith. A: From an adult Microtus pennsylvanicus. The midline septum (not shown) is to the right and dorsal is above the VNO. B: From an adult human. The midline septum (not shown) is on the right and superior is above the VNO. Scale bar = 100 μm. L, lumen rfe, receptor (bipolar cell)-free epithelium asterisk, neuroepithelium.

At the anatomical level, bipolar receptor cells can be found within the VNO of the developing human fetus (Boehm and Gasser, 1993 Boehm et al., 1994 ), but are absent in the adult (Fig. 2). Although these VNO-associated neurons appear to connect with the brain early in development, they degenerate shortly after other neurons that contain gonadotropin-releasing hormone (GnRH), presumably members of the terminal nerve (Wirsig-Wiechmann, 2001 ), migrate along these vomeronasal nerves from their origins in the olfactory placode/VNO to their destination in the basal forebrain (Wray, 2002 ). Within the brain, vomeronasal nerves normally terminate within the accessory olfactory bulb (AOB), a structure that is embedded within the main olfactory bulb but has no direct connections with it. In adult humans, the AOB cannot be located (Meisami and Bhatnagar, 1998 ).

With respect to the human VNO, the anatomical literature reveals an emerging consensus. Jacobson ( 1811 ), now known to be incorrect, states that “man is the only terrestrial mammal in which this organ is totally absent” Boehm and Gasser ( 1993 ), in their study of the fetal VNO, report that they “did not observe receptor-like cells” in the oldest fetuses and in a follow-up study, Boehm et al. ( 1994 ) state that “the vomeronasal nerve disappears … leaving only a vestigial structure in the nasal septum.” Trotier et al. ( 2000 ) are quite firm at one point that “the vomeronasal structure does not function as a sensory organ in adult humans.” Hence, any pheromone response by humans is likely mediated via the olfactory neuroepithelium rather than by the VNO.

Pheromone Response: Primers, Signalers, Modulators, and Releasers

Among primer effects in humans, those most often discussed are the effects of chemical signals on the menstrual cycle or its underlying hormonal systems (Table 3). In humans, there are many examples of signaling pheromones, including recognition of kin, gender, sexual orientation, and, at least for the MHC, genetic identity by chemical signals. Also included in this category are signals indicative of diet and disease. As an organizing construct, the modulator pheromone is a latecomer, having been recently introduced in Jacob and McClintock ( 2000 ) and McClintock ( 2000 ). These cues, originally construed as signaler pheromones (which remains possible), are thought to modify extant moods or emotional states. Although the fourth category of pheromones is the most discussed, at least in the lay literature and among the media, little solid evidence for releaser pheromones in adults can be found within the biomedical literature.

Type of pheromone Effect References for human responses
Primer Endocrine/neuroendocrine Weller and Weller ( 1993 ) Stern and McClintock ( 1998 ) Preti et al. ( 2003 )
Releaser Behavioral Varendi and Porter ( 2001 )
Signaler Informational Cernoch and Porter ( 1985 ) Jacob et al. ( 2002 )
Modulator Influences mood or emotion Chen and Haviland-Jones ( 1999 , 2000 ) Jacob et al. ( 2000 ) Ackerl et al. ( 2002 ) Preti et al. ( 2003 )

Primer pheromones.

Excellent examples of primer pheromones have been described for both male and female nonhuman animals (Halpern and Martínez-Marcos, 2003 ). The effects are numerous. Beginning early in life, exposure to chemical signals from adults of the opposite sex typically will advance the onset of puberty, while exposure to analogous signals from the same sex will retard the onset of puberty (Bronson and Macmillan, 1983 ). Estrous cyclicity in females can be radically affected by primer pheromones. Female mice living in a densely packed cage alter the composition of their urine such that it inhibits cyclicity among the females (van der Lee and Boot, 1955 ). Furthermore, exposing an isolated female mouse to bedding laden with chemical cues from the group of female mice will inhibit cyclicity in the isolate (Drickamer, 1974 ). Alternatively, adding urine from an adult male mouse to the cage of the group-housed females will disrupt the shared cessation of cyclicity (Whitten et al., 1968 ). In many species, males that are exposed to chemical cues from novel adult females will typically exhibit a spike in luteinizing hormone (Maruniak and Bronson, 1976 ), followed by a surge in testosterone (Wysocki et al., 1983 ). In some species, pregnant females exposed to pheromones of adult males that did not impregnate the females will terminate the pregnancy by reabsorbing the fetuses (Bruce, 1959 ) or, at least in microtine rodents, prematurely deliver unviable offspring (Richmond and Stehn, 1976 ).

Among humans, the most studied phenomenon that is analogous to those noted concerns the menstrual cycle (McClintock, 1971 ). Myriad, but not unanimous, reports document menstrual synchrony among females sharing a common environment (Weller and Weller, 1993 ). Where this occurs, the effects are thought to result from exposure to pheromones from a driver female (Russell et al., 1980 ) whose cycle is thought to remain unaffected but who provides the temporal cues to synchronize the cycles of other females [(Preti et al., 1986 ) see Wilson ( 1992 ) for a critique]. Depending on the stage of the cycle of driver female, these cues appear either to accelerate or to retard the onset of ovulation in recipient females [(Stern and McClintock, 1998 ) for an alternative interpretation, see comments by Whitten ( 1999 )].

Recently, another female-female effect on the menstrual cycle has been reported. In this particular instance, however, the effect was not to synchronize but to increase variability among women. Jacob et al. ( 2004 ) reported that the odors obtained from the breasts of lactating women disrupted “the normal homeostatic regulation of cycle length” in other nulliparous women who were given the chemical signals. The effect was pronounced—variability in cycles increased threefold—and was suggested to play a role in fertility in the general population of women.

Effects on the menstrual cycle are not limited to pheromones from other females. Apparently, a cue from the underarms of males can affect the menstrual cycle, and at least a subset of the hormones that underlie the cycle. In one study, females were selected for having an aberrant cycle (either much longer or shorter than the prototypical 29.5 ± 3 days). They then received an extract of secretions collected from the underarms of male donors or a control extract. When compared with the results obtained from the control group, females receiving the males' extract had a more regular cycle (Cutler et al., 1986 ).

The results of a more recent study provide a possible mechanism to support pheromone-mediated shifts in the menstrual cycle (Preti et al., 2003 ). In this study, female subjects in the first 7 days of their cycle were confined to a hospital setting and had an in-dwelling catheter inserted to collect venous blood every 10 min. In a crossover design, every 2 hr each woman received on the upper lip either an extract of underarm secretions from donor males or a control solution (phase 1). After 6 hr, the conditions were reversed (phase 2). During extract exposure, the onset of the next peak of luteinizing hormone (LH) was advanced by ∼ 20% after application of the male pheromones(s) relative to the LH response in the control condition (Fig. 3) (Preti et al., 2003 ). Across subjects, the effect was robust the pulse after pheromone application, relative to the control condition, was retarded in only 1 of the 18 subjects (Fig. 4). Although the phase of the study revealed an anticipated diurnal effect, it did not influence the effects of the males' pheromone(s).

Average latency to the next LH peak subsequent to the application of male axillary extracts (extract 47 ± 5 min), applied three times, spaced by 2 hr each, or subsequent to the application of the control solutions (control 59 ± 5 min), also spaced by 2 hr each (Preti et al., 2003 ). In an analysis of variance, the main effect of stimulus type on latency to the next pulse was significant (F(1,16) = 28.34 P < 0.001).

Average latency difference (in min) generated by subtracting the average latency to the next LH peak subsequent to the application of the control stimulus from the average latency to the next LH peak subsequent to the application of male axillary extracts (Preti et al., 2003 ). Sixteen of the 18 women had an average latency to the next LH pulse that was shorter in the extract condition than in the control condition 1 woman had latencies that were equivalent in both conditions (Wilcoxon signed rank test = 3.54 two-tailed P < 0.0001).

Signaler pheromones.

Much literature supports the claim that nonhuman animals can recognize kin via odor signatures (Wyatt, 2003 ). Not to be outdone, human mothers of newborn babies can also recognize their offspring by odor alone (Kaitz et al., 1987 ) fathers, however, fail at the task. Early exposure to chemical cues circulating in the mother's bloodstream (and thereby stimulating the olfactory epithelium) may be a possible explanation (Beauchamp et al., 1995 ). The odorprint, while modified by diet, disease, and other environmental factors (Mennella and Beauchamp, 1991 ), has in part a genetic basis. Genes within the MHC (HLA in humans) confer on an individual a unique odor that is predictive of subtle, perhaps even single gene, differences across individual genotypes (Bard et al., 2000 ). These odorprint signatures can be discriminated by scent alone (Yamazaki et al., 2000 ) and have been implicated in or suggested to influence mate choice in some species (Beauchamp et al., 1985 ), including humans (Jacob et al., 2002 ).

Much other information can be obtained from signaling pheromones. Herein lies one of the problems with the broad definition of pheromone. Is information per se actually a pheromone? For example, an urbanite has a choice of one of two stairwells into a subway system, but one of them has been scented by an earlier human visitor who was ill and vomited (a chemical cue from a member of the same species). The urbanite detects the odor and chooses the unscented entrance, thereby producing a behavioral response to a conspecific chemical cue. The result may benefit the recipient of the chemical message, e.g., prevent a stimulus-induced, perhaps retching, response. Would an independent naive observer record pheromone-mediated behavior?

Modulator pheromones.

This newest addition to the pheromone family was introduced in 2000 by McClintock's laboratory (Jacob and McClintock, 2000 McClintock, 2000 ). Modulator pheromones were purported to affect moods or emotions. Indeed, the authors state that a purported pheromone “appears to modulate affect” to elicit noted changes “rather than [by] releasing stereotyped behaviors” (Jacob and McClintock, 2000 : p. 57).

There are reports that the odor of a body changes with emotional state (Chen and Haviland-Jones, 1999 , 2000 Ackerl et al., 2002 ). People who were placed in situations that provoked anxiety, e.g., watching fear-inducing film clips, changed their body odor. These body odors were different from those collected during unprovoked conditions or when the same individuals were exposed to film clips of comedic situations. Other people were able to discriminate the differences among the various emotion-inducing conditions however, what was not reported was whether the moods of the volunteers who were sniffing the body odors were affected by the body odors that they were evaluating. Did the mood of the evaluators shift to match that of the donor?

In a much different experimental design, Preti et al. ( 2003 ) noted that an extract of sweat, collected from pads that were worn in the armpit of male donors, was able to shift the mood of females who had the extract applied to the upper lip. In a crossover design, the females were “more relaxed” and “less tense” during a 6-hr period when sweat from males was present on the lip than in the 6-hr control condition when only the vehicle was on the lip. These results suggest that modulator responses may indeed occur among humans, but much more research on this topic needs to be performed.

Releaser pheromones.

Of the classes of pheromones, releasers are most often associated with sexual attraction. This has in part a historical foundation. The pivotal publication by Karlson and Lüscher ( 1959 ) described the upwind-seeking behavior of male moths in the presence of a sexual attractant isolated from female moths. Releaser pheromones, however, exist in many more flavors and elicit various behaviors: aggression from males (Maruniak et al., 1986 ) and females (Bean and Wysocki, 1989 ) maternal behavior (Del Cerro, 1998 ), even from nulliparous females (Saito et al., 1998 ) suckling in infant rabbits (Schaal et al., 2003 ). Indeed, among humans, infants are attracted to breast odors of their mother and move in the direction of the odors (Varendi and Porter, 2001 ). To date, this crawling movement by infants is likely the only human releaser pheromone response documented in the biomedical literature.


Study participants

The study was carried out at the Department of Biological and Environmental Science of the University of Jyväskylä in Finland. The participants were all volunteers and mainly students in biology and psychology. Eighty-two women wore a T-shirt for two consecutive nights directly on the skin, after which 31 male and 12 female raters rated the sexual attractiveness and intensity of the shirt's odors. As the possible attractiveness of women's body odors might have a hormonal basis, we selected both users and nonusers of oral contraceptive pills for the study. The participants were told that the purpose of the study was to investigate whether odors affect human sexual selection, but they were not informed of the exact hypothesis. The odor ratings were carried out in three trials during three consecutive weeks in March 1999. Each trial consisted of new T-shirts wearers (26, 28, and 28 wearers randomly assigned per week, respectively), whereas the T-shirt raters were always the same. The three trials did not differ in respect to the cycle length, day of menstrual cycle, or age of T-shirt wearers (one-way ANOVA, p for all >.05). Therefore, study week was not used as a separate factor in the statistical analyses.

Collection of women's body odors

As in many previous human odor studies, body odors of women were collected by T-shirts (see Gangestad and Thornhill, 1998 Rikowski and Grammer, 1999 Singh and Bronstad, 2001 Thornhill and Gangestad, 1999 Wedekind and Füri, 1997). The unworn white cotton T-shirts were prepared by washing them with nonperfumed soap powder and keeping them in odorless plastic freezing bags after drying. Each woman received one T-shirt, a package of soap powder to wash her bedclothes before the experiment, a perfume-free soap for personal hygiene, and odorless liquid soap for hair cleaning. Women were informed about the T-shirt experiment procedure, and they were provided detailed instructions of behavioral restrictions to avoid disturbing scents. The instructions included refraining from (1) using perfumes, perfumed deodorants, and perfumed soap powder (2) eating odor-producing food such as garlic, onion, strong spices, herbs, cabbage, celery, asparagus, yogurt, and lamb (3) smoking cigarettes, drinking alcohol, and using drugs and (4) sleeping with another human and sexual activity. When a woman did not wear her shirt, she stored it in an odorless freezing bag. The women returned their shirts in the freezing bags in the second morning between 0800–1000 h, and they were asked to honestly report possible violations of the instructions. One woman reported that she had not followed our instructions, and her shirt was excluded from the study. Women were also asked whether they use contraceptive pills, and to report the first date of their last menstrual bleeding and their mean cycle length. These enabled us to calculate on which day of the menstrual cycle the experiment had taken place.

There was no difference in the age of participants between normally ovulating women (mean = 23.2, range = 16–49, SE = 0.78, n = 41) and pill-using women (mean = 22.5, range = 17–32, SE = 0.48, n = 39, two-sample t test: t = −0.73, df = 78, p >.5). Use of contraceptive pills changed the cycle length of women (nonusers: mean = 30.1 days, range = 25–42, SE = 0.53, n = 42 pill users: mean = 28.1 days, range = 24–30, SE = 0.14, n = 39, two-sample t test: t = −3.57, df = 46.5, p <.001). When we analyzed the sexual attractiveness and intensity of odors in relation to menstrual cycle, every woman's day of menstrual cycle was corrected by her cycle length with an equation: [(28/cycle length) × day of menstrual cycle].

Odor rating sessions

The odor rating sessions were arranged at the same day the women returned their T-shirts. The shirts were conserved in 4-l glass jars, which were labeled and sealed. In addition to the shirts worn by women, three clean shirts that had not been worn were included in the sample (one shirt per week). The participants and supervising researchers did not know who had worn the T-shirts or other information about the wearers. The participants sat at tables while the glass jars were randomly circulated between the tables. During a rating procedure, a participant opened a jar and smelled a shirt by holding it beneath his or her nose. Then he or she rated the odors of the shirt for sexual attractiveness (range, 1–10: 5 = neutral, 10 = highest) and intensity (range, 1–10) and wrote the ratings on a questionnaire. After this, he or she closed the jar and passed it to the next rater. To measure the repeatabilities (see below), a second rating session with changed labels and randomized order of shirts was arranged. The sessions lasted approximately for half an hour with a 15-min break between them.


The repeatabilities of shirt ratings were calculated from the one-way ANOVA comparing T-shirts over the both rating sessions (see Lessells and Boag, 1987). The repeatabilities (R) were as follows: for attractiveness (males: R =.85, ANOVA F83,2399 = 12.613, p <.001 females: R =.62, ANOVA F83,814 = 4.252, p <.001) and for intensity (males: R =.74, ANOVA F83,2398 = 6.629, p <.001 females: R =.47, ANOVA F83,813 = 2.803, p <.001).

Statistical analyses

The correlations of ratings between the two rating sessions were high (attractiveness, males: r =.91, p <.001, n = 84 females: r =.82, p <.001, n = 84 intensity, males: r =.91, p <.001, n = 84 females: r =.81, p <.001, n = 84). Therefore, mean attractiveness and intensity ratings of the first and the second rating sessions for each rater were used when analyzing the odor characteristics in relation to menstrual cycle. There was no statistically significant correlation between sexual attractiveness and intensity of the odors (males: r =.10, p =.367, n = 84 females: r =.03, p =.779, n = 84). Consequently, we treated these as separate variables in all the analyses.

We analyzed the attractiveness and intensity of body odors along menstrual cycle with a linear mixed model specified for the mean ratings of the T-shirts. In the first stage of analysis, the model contained all the effects involved in the experimental design. That is, the main effects of the sex of the rater (SEX) and the use of contraceptive pills of the wearer (PILL), as well as their interaction (SEX × PILL), were included as fixed effects. Because the same T-shirts were smelled by both male and female raters, the shirt effect was included in the model as a random effect nested within the pill effect. Thus, the design is similar to the repeated-measures design with the T-shirts as subjects, PILL as a between-subjects factor, and SEX as a within-subject factor. We also wanted to examine the effect of the day of menstrual cycle on the responses. Within the menstrual cycle, this effect was expected to be of quadratic form: the levels of attractiveness and intensity are highest in the ovulatory phase (middle of the cycle) and decrease toward the beginning and end of the cycle. Because the preliminary data analyses seemed to support this hypothesis, we added the linear (DAY) and quadratic (DAY 2 ) effects of the day into the model as covariates. No evidence on higher-order effects was found. Finally, we added the two-way and three-way interactions of the covariates with PILL and SEX to account for the possible variation of the day effect over the four PILL × SEX groups.

This full model (in the sense that it contains all possible interactions in the design) was first estimated and evaluated. Then we hierarchically simplified the model as far as possible by removing the nonsignificant effects one by one, starting from the most complex least significant interactions. The model that could not be simplified any more without dropping a significant effect or violating the hierarchy principle (i.e., nonsignificant lower-order effects cannot be removed if a significant higher-order interaction of the same factors is present) was selected as the final one.

The model was built in this stepwise way independently for both attractiveness and intensity. In each step the estimation and significance testing was carried out by the MIXED procedure of SAS software ( SAS Institute, 1999b), using the restricted maximum likelihood (REML) method ( Patterson and Thompson, 1971) with related F tests. In our case these agree with the usual F tests of the repeated-measures ANOVA. The degrees of freedom for the F tests were calculated by the method of Kenward and Roger (1997).

The estimates of the fixed effects in the final model were used in calculating the estimated second-order polynomial regression of the responses on the day of menstrual cycle in each PILL × SEX group. These calculations were performed by the SAS/IML software ( SAS Institute, 1999a).


Since the beginning of the human existence on the earth reproductive biology remained a main concern of research because of its importance. It is widely recognized and demonstrated that odors play an important role in mammalian reproduction. A large number of studies have been carried out in humans, in order to investigate possible pheromones, their properties, mechanism of action, and possible receptors for their action. Till now scientific studies indicated that humans use olfactory communication and are even able to produce and perceive certain pheromones. This review article aims to highlight the role of human pheromones as aphrodisiacs

Sharpened olfactory temporal resolution related to high-speed flight maneuvers

The challenge for insects is to sample the odor strands as frequently as possible, as well as to sample the inter-strand pockets to make sub-second, in-flight decisions about maneuvering in the wind flow, whose direction of movement provides the moth with the only information available about the toward-source direction. Failure to perform these feats of olfactory temporal acuity that, as Lei et al. [2] have shown, are linked to proper wind-steering maneuvers, can mean failure to find a mate before competitors do. Not responding rapidly enough to contact with a strand can result in lack of progress straight upwind to the source. Failure to rapidly respond to a pocket of clean air between strands by not immediately stopping upwind progress and initiating side-to-side crosswind 'casting' flight can result in erroneous steering, both off-line from the toward-source direction as well as away from the direction to which the plume has swung in a shifting wind field [5].

The insect signal acquisition and processing system for sex pheromone starts with neuronal inputs from the tens of thousands of ORNs on the male antenna, each ORN being differentially and tightly tuned to only one of the two or three components that comprise that species' blend of sex pheromones. Axons from each of these classes of pheromone-component-tuned ORNs travel to the antennal lobe of the brain at the base of the antenna and arborize in their own class-specific knot of neuropil called a glomerulus, which resides there within a cluster of other pheromone-component-specific glomeruli called the macroglomerular complex (MGC Figure 1). It is here that the first postsynaptic interneurons, called local interneurons, impose GABA-related inhibition on neurons in neighboring MGC glomeruli.

Frontal view of the face of a male Helicoverpa zea moth showing the two antennal lobes at the bases of the antennae. The preparation has been histologically cleared so that the many antennal lobe glomeruli are visible as spheroidal shapes. Asterisks denote 'ordinary' glomeruli that receive inputs from antennal neurons responding to general environmental odorants such as plant volatiles. The ordinary glomeruli reside in a large cluster in each antennal lobe (Ord). Larger glomeruli that receive inputs from pheromone component-tuned neurons on the antenna reside in their own cluster called the macroglomerular complex (MGC) and are labeled with an 'm'. Ant, the remaining bases of the antennae and antennal nerves eye, optic lobe.

This form of olfactory lateral inhibition has been implicated in enhancing the contrast between the activities across the ensemble of glomeruli to produce a contrast-enhanced relative pattern of outputs across the array of different projection interneurons exiting the various glomeruli and projecting out to the mushroom bodies and the lateral protocerebrum (Figure 2). The across-ensemble pattern of projection interneuron activity results in a representation of pheromone blend quality as a spatial pattern in the mushroom body. An earlier study [6] demonstrated the effects of a GABA blocker, picrotoxin, on odor-space discrimination. Impairing the activities of GABA-ergic neurons and dampening local field potential oscillations (believed to be set up by interactions between the antennal lobe and mushroom bodies) reduced fine-grained odor-quality discrimination by honey bees.

Top view of the head of a Helicoverpa zea male moth stained histologically to highlight the regions of the male moth brain involved with pheromone and other odorant signal processing and odor-quality discrimination. The anterior face of the moth is looking up toward the top of the figure. Sex pheromone information comes into the antennal lobe glomeruli of the macroglomerular complex (MGC) from the antenna. General odorant information comes from the antenna into the ordinary glomeruli (Ord) of the antennal lobe. Inhibitory GABA-ergic local interneurons form a network cross-linking all the antennal lobe glomeruli and help shape the relative levels of excitation emerging from each glomerulus via projection interneurons. The axons of these projection interneurons project in a single tract to the back of the brain to synapse first with neuropil in the mushroom body (MB) before continuing on to synapse with neurons in the lateral protocerebrum (LP). Axons of other projection neurons that also carry relative levels of excitation from antennal lobe glomeruli project in a second, different tract directly to the LP, bypassing the MB. The LP is where behavior-initiating descending interneurons synapse to send command signals to motor centers. Adapted from Lee et al. [11].

Lei et al. have now demonstrated the importance of lateral inhibition in the temporal domain of olfactory acuity. They used bicuculline methiodide to block the activity of GABAA inhibitory pathways in the pheromone-related glomeruli of the moth MGC and showed that these pathways work to silence neuronal firing of projection interneurons in the clean-air pockets between pheromone strands. Impairing the GABA-ergic neurons did not affect peak firing in response to pheromone strands, so the significant reduction in projection neuron firing between strand-induced bursts helps improve temporal resolution and accentuate the variations in pheromone flux.

Notably, Lei et al. directly linked impairment of the temporal contrast-enhancement circuitry in the antennal lobe with impaired upwind flight behavior of male moths. They thus demonstrated the importance of temporal pheromone strand resolution by the inhibitory antennal lobe circuits to successful pheromone source location by flying moths. Researchers decades earlier had demonstrated the importance of pheromone flux variations to successful upwind flight behavior by manipulating the pheromone plume flux itself and not the olfactory pathways, as Lei et al. have done in their current study.

After RH Wright [7] first pointed out that odor plumes are composed of small strands of highly concentrated odor that might be important in influencing insect behavior, subsequent studies showed that flux change, that is, pheromone intermittency, is crucial for successful upwind flight by males. Presentation of otherwise attractive pheromone odors as a uniform fog or cloud caused no upwind flight, just side-to-side cross-wind casting flight [8]. When such clouds were pulsed and interspersed with clean air at a frequency of 1 or 2 Hz, upwind flight proceeded successfully [9]. Further experiments suggested that individual strands within a plume could evoke upwind flight behavior, and experimentally generated single strands were shown to promote single upwind flight 'surges' within approximately 0.3 seconds after strand contact (see [5, 10] and references therein). Equally fast reaction times to pockets of clean air were suggested to be as behaviorally important for successful and rapid source location as the reaction to the strands themselves hence, the selection over evolutionary time for high-fidelity flux resolution in moth pheromone olfactory systems [5].

Lei et al. have convincingly demonstrated the importance of inhibitory GABAA-ergic circuitry in preserving a high-fidelity temporal representation of pheromone flux in projection interneurons deep within moths' pheromone olfactory pathways. Previously known to be important for optimizing odor quality discrimination, GABA-ergic interneurons have now been shown to be behaviorally important enhancers of temporal olfactory acuity. Some types of projection interneurons arborize first in the mushroom body and then in the lateral protocerebrum (Figure 2), where synapses with behavior-generating descending interneurons occur. Another type projects directly to the lateral protocerebrum, bypassing the mushroom body. It seems possible that because there are two distinct odor-resolution systems in insect olfaction, one for high-fidelity representation of odor space and another for high-fidelity reporting of odor time, moths may use these two different pathways in the brain that have been selected over evolutionary time for different, but complementary, behavioral purposes.

The Strange Science Of Sexual Attraction

Attraction, like romantic love, works in mysterious ways.

While we'd like to think that we know why a particular person catches our eye, there are a number of invisible forces at work that determine which members of the opposite sex we become interested in -- and which ones we don't.

Of course, there are a number of factors that go into who we choose to be with, including personality traits, interests and values and physical appearance. But when it comes to immediate, knee-jerk physical attraction, we often can't pinpoint why exactly we're drawn to someone. Even as scientific research has shed more light on the factors that contribute to our selection of a sexual mate, the biology of attraction is complex and not yet fully understood -- and it doesn't help that attraction is particularly difficult to replicate in a lab.

So what really is happening when the sight of a hot guy or girl makes us instantly swoon? Human biology and evolutionary psychology has some answers.

Here are some of the subtle but powerful factors that may help determine who we're attracted to.

We fall in love at first "smell."

"Smell" is the woefully inadequate way we describe sensing someone's pheromones -- a type of scent-bearing chemical secreted in sweat and other bodily fluids. Pheromones are known to be involved in sexual attraction in animals, and research suggests that they may also play a role for people. A type of pheromone called a "releaser" -- which includes the compounds androstenone, androstadienone and androstenol -- may be involved in sexual attraction, according to a Reactions: Everyday Chemistry video.

"We've just started to understand that there is communication below the level of consciousness," psychologist Bettina Pause, who studies pheromones, told Scientific American. "My guess is that a lot of our communication is influenced by chemosignals."

In one study, female participants were tasked with the unpleasant directive to smell men's sweaty undershirts. The researchers found that women could smell how symmetrical a man was, and using that information, judged his attractiveness. (In both men and women, symmetry is known to be an important factor in attractiveness.)

Men can detect a fertile woman.

Men can actually sense fertility on a woman, perhaps due in part to her pheromones. During the most fertile time in her menstrual cycle, a woman gives off a different scent which may make her more attractive to potential male suitors. Research from the University of Texas at Austin investigated this phenomenon by asking a group of women to wear T-shirts to sleep during both fertile and infertile points in their cycles, and then asked men to smell the T-shirts and assess which ones they found most pleasing. Overwhelmingly, they judged the shirts worn by the fertile women to be more "pleasant" and "sexy."

A woman's face may also appear more attractive to men during the most fertile point in her cycle. A British study conducted in 2004 asked a group of 125 men to look at two pictures of the same woman, at times of high and low fertility in her cycle, and to assess which photo was more attractive. Nearly 60 percent of the men rated the photos of the women's faces at peak fertility (eight to 14 days after her last period) to be more attractive.

The sound of a woman's voice also plays into a man's judgements of a woman's attractiveness. A recent study found that a woman's voice sounds most seductive at the most fertile point in her menstrual cycle -- and that hearing a woman's peak-fertility voice can literally make a man's skin tingle.

Women quickly assess markers of masculinity.

A large body of evolutionary psychology research has shown that, in general, women tend to prefer more masculine-looking men -- perhaps because masculine features like broad shoulders or a strong jawline are indicators of virility and good health. But today, this doesn't always hold true.

Women may have evolved to seek out virility, but that doesn't mean that their preference in a modern context is always for "manly" men (and ditto for men's attraction to "fertile looking" women). Not all -- or even the majority -- of women prefer more masculine men. One study found that context matters: Women living in poorer environments may have a greater preference for masculine men, but women in more developed areas prefer more feminine-looking men, according to a study from the Face Research Laboratory.

"From an evolutionary perspective, masculinity is basically man's way of advertising good genes, dominance and likelihood to father healthier kids," the Wall Street Journal explained. "When disease is a real threat, as it had been—and arguably still is—heritable health is invaluable."

One time this preference may hold true is when a woman is at the most fertile point in her cycle. One study found that women whose partners had less masculine facial features reported attraction to more masculine-looking men when they were ovulating. However, women whose partners had more masculine features did not report the same eye-wandering. However, these findings only applied to women in short-term relationships -- not serious, committed partnerships.

The Pill might change a woman's preference in men.

Is she really attracted to you -- or is it just her birth control? A number of studies have suggested that hormonal contraception may have some effect on women's preferences for sexual partners.

A man's smell provides a woman with information about his major histocompatibility complex (MHC) genes, which play an important role in immune system function. As the thinking goes, women prefer men whose MHC genes differ from their own because children with more varied MHC profiles are more likely to have healthy immune systems -- which makes a whole lot of sense from an evolutionary perspective. However, research has shown that women on the pill actually display a preference for men with more similar MHC genes to their own. Scientists aren't entirely sure why this happens, but one hypothesis is that the hormonal changes involved in pregnancy (which the Pill mimics) might draw women more to "nurturing relatives."

Even within long-term committed relationships, changes in hormonal contraception use might affect a woman's sexual satisfaction with her male partner. "Women who had met their partner while taking the pill and were still currently taking it -- as well as those who had never used the pill at any point -- reported greater sexual satisfaction than those women who had begun or stopped using the pill during the course of the relationship," lead researcher Dr. Craig Roberts said in a statement.

But personality is important, too.

It's not all about a person's looks and their chemical makeup -- certain personal characteristics can also play a role in determining how "hot or not" someone is.

Kindness, for instance, can make a person more attractive in addition to making them more likable. A 2014 study found that positive personality traits actually increase perceived facial attractiveness. The researchers asked 120 participants to rate 60 photographs of female faces in neutral expressions. Two weeks later, they were asked to evaluate the same photos, but this time, half of the photos were accompanied by positive personality descriptors like kind and honest, and half of which were accompanied by negative descriptions like mean and dishonest. A control group saw the photos without any descriptions.

The photographs with the positive personality descriptions received the highest ratings for facial attractiveness, while the group with negative descriptions was ranked as less attractive than both the negative and the control group.

"We find that 'what is good is beautiful,' with personality reflecting desired traits as facial attractiveness," the researchers wrote. "This phenomenon can also be called the 'halo effect.' We can thus presume that personality traits may contribute to judging facial attractiveness and that the personality traits desired in a person are reflected in facial preference."

Who we're attracted to is still a very individual matter.

While there is something of a science to the romantic and sexual partners we choose, at the end of the day, attraction is still completely unique to each of our individual makeups and preferences.

Anthropologist Helen Fisher, who has studied love and dating extensively, explains that we each have individual "love maps" that determine who we gravitate towards.

"These love maps vary from one individual to the next. Some people get turned on by a business suit or a doctor's uniform, by big breasts, small feet, or a vivacious laugh," Fisher writes in Psychology Today, adding, "But averageness still wins."

Fisher cites a study in which participants selected faces of 32 women, and used a computer program to make their features look more average. Then, they showed these photos as well as 94 photos of real female faces to a group of college students. Only four of the photographs of real female faces were rated as more attractive than the "averaged" faces.

As Fisher suggests, while individuals and cultures have their own standards for what they consider attractive, there are some fairly universal qualities that we all look for, including a clear complexion, symmetrical faces, wide hips (for women), and a general appearance of health and cleanliness.