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So, what's happening specifically regarding the detection of pheromones?
What are the systems that detect pheromones, how is this information processed
within the brain and how are behaviors, specific behaviors, generated?
How does an animal know that the signal comes and leads to fighting behavior
or mating behavior? How is the quality of the pheromonal information received,
perceived and how does it lead to behavioral changes?
As I mentioned just before, there are these two systems,
the vomeronasal system on one hand, the olfactory system on the other hand,
and the assumption for a very long time
was that the vomeronasal system was specialized into the detection of pheromones
and the olfactory system was specialized in the detection of odorant chemicals.
And this notion came from surgical ablation experiments
in which people had surgically ablated the olfactory epithelium
and this led to impairment of odorant detection,
or surgical ablation of the vomeronasal organ and this led to defect in mating or aggressive behavior
and therefore, presumably the detection of pheromones.
So, surgical experiments, surgical ablation, were a big cue
into the role of each of these two separate systems
and also the notion that these two systems share the work
between cognitive smell and instinctive smell
also originates from the study of the central projection of this system.
So, the olfactory epithelium is connected to the main olfactory bulb,
and in turn, to a number of nuclei in the brain
that are together forming what is called the primary olfactory cortex
and then the information is distributed very widely within cortical and neocortical areas of the brain
and therefore leads to this cognitive perception of a smell.
And in contrast, information that is detected by the vomeronasal organ
seems to be processed by an entirely different and independent central pathway
from the vomeronasal organ to accessory olfactory bulb
to then specific areas of the medial amygdala in the limbic system
that are themselves connected to specific areas of the hypothalamus
that are specialized in aggressive, and triggering aggressive and mating behaviors.
So, specialized reproduction and social behavior in general.
So, it seems, therefore, to make sense that these areas that are involve
the primary olfactory cortex and then higher cortical areas
would indeed be responsible for the cognitive detection of smell
whereas areas of the brain that are part more of the limbic system,
the amygdala and the hypothalamus, are more involved in processing pheromonal signals
and the trigger of reproductive and aggressive behavior.
So, this seems all very logical, and at the molecular level,
it was also very interesting to discover that neurons of the main olfactory epithelium
seem, through a set of channels that are cyclic nucleotide gated,
therefore, the signal transduction of olfactory signals use cyclic nucleotides
that in turn lead to the opening of ion channels
and therefore, enable the translation of the binding of odorant to the receptor
into an electrical signal, a change in membrane potential.
And in contrast, in the vomeronasal organ,
we don't find any functional cyclic nucleotide gated channels,
what we found several years ago in collaboration with Emily Liman and David Corey,
is the very strong and specific expression of a distinct ion channel called TRPC2,
that is again very highly and specifically expressed in vomeronasal organ
and is responsible for the VNO signal transduction.
So, at the molecular level we have these two ion channels
that are each essential for olfactory transduction and vomeronasal transduction
and these therefore provide a terrific genetic tools to investigate,
or reinvestigate, if you wish, the function of each of these two sensory pathways in the brain.
So, by genetic manipulation of the gene encoding the TRPC2 channel,
we perform a knockout of the TRPC2 channel and therefore, led to an animal,
generated a line of genetically modified mouse, in which the VNO does not function,
because the TRPC2 channel is non-functional, is mutated,
and therefore the entire vomeronasal pathway is made non-functional.
And therefore, this mutated animal, this mutant,
doesn't have a functional vomeronasal organ, is unable to detect pheromones,
and we can therefore investigate the physiological role of the vomeronasal organ
in the animal physiology and behavior.
And similarly, but looking at the knockout of the cyclic-nucleotide gated channel,
we and others have been able to investigate the behavioral function
of the main olfactory system.
So, this is just to show you the expression of this TRPC2 ion channel.
What you see on this part of the slide is a section
through this tubular structure that is formed by the vomeronasal organ
and what you can see here is the neural epithelium that borders the lumen
through which the pheromones are, rise in contact to neurons.
And in red is immunostaining with the TRPC2 channel
and you can see that the protein is highly expressed and very specifically expressed
along the sensory terminal of the VNO neurons,
here seen even better, on a dissociated neuronal preparation,
you can see the sensory dendrite here, where the sensory, the receptors and the channels are,
so, you know, these expression patterns really suggest an important role of the TRPC2 channel
in the sensory transduction in the vomeronasal organ.
And the idea is that the two families of epithelial?? pheromone receptors, V1Rs and V2Rs,
when binding to pheromonal signal, lead to a signal transduction cascade
and in turn, to the opening of the TRPC2 channels.
So, by the knockout of the TRPC2 channel, one can completely abolish the signal transduction
and lead to an animal without a functional vomeronasal organ.
So, this is what we did, and the first experiment that we performed
when we obtained a mutant animal is to indeed validate the claim that the TRPC2 channel
is essential for VNO signal transduction.
And the experiment, so here is just the demonstration that in the TRPC2 mutant
there is no TRPC2 protein made anymore, compared to ubiquitous proteins, such as beta tubulin,
and in collaboration with Markus Meister, an actual physiologist in my department,
and Tim Holy, who was a post-doc in Marcus's lab,
we performed the electrical recording of the VNO neurons
in response to pheromonal stimuli.
So, the idea is to use a flat electrode array
in which each of the dots here represent a different electrode that can record from neurons
the electrical activity of neurons in the vicinity
and a VNO epithelium is pressed flat against this electrode array
and maintained by a mesh and then we can puff pheromonal stimuli
and record the activity of the neurons that have been stimulated by specific chemical cues.
And when we did the experiment and compared the situation
in the wild-type animal or the heterozygous animal to the situation in the mutant,
it became very clear that pheromonal stimuli leads to an increase in the spiking rate
of VNO neurons, are recorded by the electrode array,
but there was absolutely no stimulation in the TRPC2 mutant.
So, in other words, the VNO neurons are unable to respond to pheromonal stimuli.
We know that there are neurons here because if we stimulate the preparation with potassium chloride,
high concentration of potassium chloride,
we can see a very strong, non-specific neuronal firing that comes just from the depolarization of the cells;
however, these cells are unable to specifically respond to pheromonal stimuli.
So, the VNO is basically silent and what we have is a mouse line
in which the olfactory detection can occur, but the vomeronasal detection is completely impaired.
So, what's happening to the behavior of these animals?
Well, to our big disappointment at first, this animal didn't seem to show any phenotype.
We expected, from surgical experiment, that animals without VNO
would be unable to mate. But, when we put a male mouse in the presence with a female,
a male mutant, in the presence of a female, they were mating perfectly normally,
in fact, exactly with the same frequency as wild-type animals.
So, we were very disappointed and even questioned what really,
what the vomeronasal organ good for?
And then we thought a little bit further and decided to study another set of behavior,
and we used very well known observation from Konrad Lorenz
that described behavior along these words.
If you put together, into the same container,
two sticklebacks, lizards, robins, rats, monkeys or boys,
who have not had any previous experience with each other, they will fight.
You can add to these two politicians, two scientists, two whatever,
when you put two males of any animal species in the same cage or room,
they will tend to fight with each other.
Well, we did this experiment
and together with the wild-type male mouse
and mutant male mice, and I'm going to show to you
the behavior or the mutant compared to that of the wild-type.
The behavioral paradigm that we use is as follows.
We know that in rodents, fighting behavior between two males
arises from the detection of male pheromones.
So, in order to set up an experimental system in which we can control the presence
or not of the male pheromones that trigger the male behavior,
our paradigm was chosen as follows.
We had a resident male, so an animal that stays in its cage for a couple of weeks,
and kind of established its territory, and we then introduced into that resident cage
an intruder. And the intruder is of different kind.
We first introduced a male intruder that is a castrated male.
Pheromones are under the control, pheromone production is under the control of testosterone,
and therefore, the castrated male is unable to produce any male pheromones.
And when we do this experiment, you can see in this video,
so you have a resident, the resident male,
and the castrated animal further here, the intruder does not emit any male pheromones,
and as you can see, these two mice just coexist very peacefully.
So, there's not even really seem to be any specific behavior of one animal versus the other.
Now, in the next video what you are going to see are the same two animals,
but the experimentator has put now 10 microliter of male pheromones
on the fur of the castrated intruder.
So, this castrated intruder does not naturally emit any pheromones,
but the pheromones, the male pheromones, is added exogenously,
and when this is done, you have now, the intruder here,
as you can see the resident detects the male pheromones
and immediately starts to fight.
So, the resident has adopted this defensive posture,
really doesn't understand what's happening to him,
and, as you can see, the resident is really a very aggressive,
and this is an extremely robust behavioral reaction,
which is a male detecting another animal emitting male pheromones will very brutally attack that animal.
Ok, so in the next, so that was the positive control,
this is what wild-type mice do, a male mouse detecting another male using olfactory cues
will attack that other males.
Now, if we use a TRPC2 male,
and so this is the male animal,
and this is the same intruder here that has been swabbed with urine,
what you see if strikingly different.
And I hope that even people without any experience in male behavior,
in mouse behavior, can very well visualize that what we have here
is absolutely not a fighting behavior, but instead a very surprising mating attempt
of the male mutant versus the other male.
So, this was extremely puzzling, extremely surprising,
the male mutant, instead of attacking the other male,
is trying to mate with it.
So, what's going on? Well, the key experiment was to actually put both male and female in the same cage.
So, again, if you have a male mutant in the presence of a female,
the mutant will mate perfectly normally,
but if you now put also a male in the cage, a male intruder,
what we discovered to our big surprise is that the mutant is unable to discriminate
between males and females and in fact, attempts to mate with each of them
with equal frequency.
And that led us to suggest, to propose, that the role of the vomeronasal organ
is not to trigger mating behavior, as was what was expected from the literature,
an animal without a functional VNO seemed clearly able to mate normally with a female,
but these animals seemed completely unable to discriminate between males and females.
And so, we even control for this behavior in a large arena
that you can see here. So, this is result from the observation that social behavior in general
can be very different in small cages or in more natural conditions.
So, we put a bunch of male mutants in the cage, and left them for several weeks,
just, you know, letting them do whatever they wanted,
for extended period of time and recording them constantly.
And as you can see, when we play the video,
is these males that form these courtship chains that are quite striking
in which one male is trying to copulate with the male in front,
and is trying to be copulated with the male just behind.
Those are extremely striking behavior that can go on for several minutes.
Now, you know, I'm showing this for entertainment value, but also for a very interesting purpose also,
which is that these courtship chains that are observed in the mouse
are actually strikingly similar to the courtship chains that have been observed in Drosophila
in a particular mutant called the fruitless mutant.
So, fruitless is a transcription factor that has many splicing variants,
some of them are sexually dimorphic, and the mutation of the male specific splicing variants
lead to these male flies that show these male-male courtship chains
that are indeed very similar to what we've observed in the TRPC2 mutant mice.
Now, this is very striking because fruitless is a transcription factor
that is expressed very widely within the brain
and is thought to be responsible for the development of the neuronal circuit
that enables courtship behavior, and the TRPC2 channel is an ion channel
expressed only in sensory neurons that give information about the gender of the animals.
And I really very clearly found the similarity of the behavior very striking.
Obviously it's a very interesting question of whether or not the mammalian brain
is expressing a fruitless equivalent,
and so far, nobody has really been able to find interesting candidates.
So, from this study, we propose a model of the control of mating behavior,
reproductive behavior, in the mouse that is quite different from the classical view
of the role of the vomeronasal system. What we found is that sensory cues
that are independent from the vomeronasal system are sufficient to trigger mating behavior.
And the role of the vomeronasal organ is to provide another type of information
which is gender identification.
So, here, there are really two systems that work with each other.
One is the vomeronasal information that provides information about gender,
and the other one, some other sensory signal that is sufficient to trigger mating behavior.
And very clearly, in the absence of vomeronasal information,
the default behavior is mating behavior.
And when a male is encountering another male,
then the vomeronasal cues detect signals that say no mating behavior,
but instead aggressive behavior.
And so, this, obviously is quite different from the classical view of the vomeronasal organ
in triggering mating behavior and I will come back later during the talk
on some possible explanation of the discrepancy between the results obtained with TRPC2 channel,
so, genetic ablation,
compared to what has been obtained with classical surgical ablation.
So, I'll tell you a little bit why I think those results were different
and this comes from results that we obtained very recently
and that I will describe in the third part of the talk.
Now, this is obviously very striking,
this shared work between vomeronasal system and other sensory cues
in the control of gender identification and at that point,
I really was curious, how are other animals distinguishing the sex of their conspecifics?
So, I went into the literature and investigated a little bit what people had described
in other species. And here is what I found.
So, in this particular species of bird, the shell parakeet,
this is a female, and this is a male.
And the animal, the parakeet, recognize the gender of their conspecific
based on the presence of these blue dots on the top of the beak.
And this particular blue dot is essential for sex identification,
which is that if you paint a blue dot on the beak of a female,
the animal now is identified as a male, and other males will attack that female with a blue dot,
thinking it is a male.
And similarly, if you mask the blue dot from the beak of a male,
then the other males are going to try to mate with this male without the blue dot,
by thinking it's a female.
So, the blue dot, the visual cues, the identification of this blue dot,
is essential to the identification of this animal as a male or a female.
Similarly, in this other species of bird, the American flicker,
here's a female, here's a male,
and what provides the identification of one being male, the other one a female,
is the presence of the black moustache.
So, if you were to mask the black moustache on the male,
this animal would be identified as a female
and the other males will try to, attempt to, copulate with this male without a moustache
and this animal here that is a female, if you plant a black moustache,
males will attack that female, thinking it's a male.
And Tinbergen, who was a very famous etiologist,
described this by calling those signs the badges of masculinity,
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.
And so this is quite interesting, the visual recognition of the gender identity,
and I think that what we found for the pheromones is the olfactory equivalent
of the badge of masculinity, which is the vomeronasal organ is responsible for
discriminating between males and females.
Now, obviously the animal we care the most about are humans.
Do human, what kind of strategy do humans use
and is the vomeronasal organ also being used for sex identification?
And here, things are likely to function very differently.
The TRPC2 channel which is responsible for the function of the vomeronasal organ
in rodents is a non-functional gene in humans
and actually in higher primates.
This little triangle that you see here, there are 9 of them,
are the site of deleterious mutations such that higher primates and humans
have a number deletion or frame shift or nonsense mutation
that make this gene unable to generate a functional protein.
And it's quite interesting to actually look throughout evolution,
when these mutations occurred. And this is this work of the laboratory of Emily Limon
that shows that the mutation really starts to accumulate
at the split between new world monkeys and old world monkeys and apes,
so all these part of the tree of higher primates really is unlikely to use the vomeronasal organ
as a tool to discriminate sex.
And I think what's quite interesting and was proposed by Emily Limon's group
is that this split here between the new world and the old world monkeys
also correspond to the duplication of the red and green opsin genes
such that animals in this part of the tree here have an additional opsin receptor gene
and therefore, the ability now to discriminate between two colors,
red and green, whereas animals in this part of the tree have one opsin gene
that detects both green and red color, and so this is being perceived as one particular color,
whereas here, those animals can discriminate between these two wavelengths of photons
that are very close to each other, but if distinguished by distinct receptors,
can now appear as distinct colors.
And this, from an evolutionary point of view
can provide an enormous advantage.
For example, the ability to discriminate between a ripe and a non-ripe fruit,
ripe fruit is full of calories, full of sweet,
and this is obviously very advantageous for animals that are able to distinguish those highly nutritious food
from a non-ripe fruit that doesn't have all this properties.
So, to come back to our two systems, we've seen using genetic mutation
that the vomeronasal system is specialized into detecting the sex identity of individuals,
individual animals, and that the dichotomy
between pheromone perception in the olfactory, in the vomeronasal system,
is not that absolute; in other words,
that mating behavior can occur without the vomeronasal organ,
therefore, there has to be something else that provides information to the animal
about the presence of conspecific and the ability to mate.
Now, how are we going to go explore the system further?
Well, as I mentioned, we and others have identified a specific receptor
for chemicals detected in the vomeronasal organ and Richard Axel and Linda Berg
discovered the olfactory receptors responsible for detection in the main olfactory system
and so, one really interesting goal is to try to understand what are the chemicals
that are detected and provide animal with information about the gender identity of the animal
or which one provides information that leads to aggressive behavior,
or any type of behavior that is triggered by these two systems.
And this direct question, what ligand generate what type of behavior, is a little bit difficult to address right now
because of technical difficulty into expressing pheromone receptors in vitro
and therefore, finding an easy, high-throughput assay to identify the ligand of these receptors.
And so, what the strategy that my lab decided to use
is start in the center of the brain, instead of the peripheral organ.
So, instead of trying to identify what are the receptors involved in specific behaviors,
and then trying to go and follow the circuitry in the brain,
we decided to do exactly the opposite, which is it well known that specific nuclei in the hypothalamus
are involved in aggressive behavior or reproductive behavior
and we therefore decided to trace the input to those specific centers,
in other words, what are the areas of the brain
and what are the specific neurons of the vomeronasal organ or olfactory epithelium
that send input, processed by different brain circuitry, that end up in, let's say,
an area of the brain involved in reproductive behavior or in aggressive behavior.
This requires two parameters.
One is the type of neurons in the brain that we want to investigate,
so, a specific set of neurons that are clearly involved in either reproduction
or the control of aggressive behavior,
and the second parameter is to find a tool that enable to link this particular set of neurons
to the connected circuit in the brain.
So, the set of neurons that we decided to study first
are neurons that express, release, express and release
a very particular neuropeptide called luteinizing hormone releasing hormone.
This is a neuropeptide that is expressed by a very small population of neurons
in the medial preoptic area in the hypothalamus.
They might be as few as six or seven hundred of these neurons
that are dispersed within this very large area of the hypothalamus,
called the medial preoptic area.
These neurons synthesize LHRH and release it in the portal vein
and the neuropeptide LHRH is absolutely essential for the control of fertility and reproduction
in vertebrates.
So, animals that are deficient in LHRH are sterile and do not develop their ***,
functional ***, and are impaired in *** behavior.
The function of these cells is as follows.
They release LHRH into the portal vein that will then interact with neurons with cells in the pituitary gland
and lead to the release of LH and FSH that is in turn is released into the blood
and lead to the development of the function of the ***,
both males and female ***.
These functional *** will in turn release steroid hormones,
the steroid hormones will, on one hand, lead to the development of the secondary *** traits
and also provide feedback to the brain in both enhancing *** behavior
but also providing a feedback to the release of LHRH.
LHRH also directly communicates with other areas of the brain
by synaptic contact and are essential for *** receptivity
and modulate *** behavior.
Now, what is quite interesting is that these neurons are really the master regulators
of reproduction and fertility in the animals
and therefore, their own function is very tightly regulated.
They are sensitive to both the internal and external state of the animal.
So, one interesting factor that controls their function
is actually a very unknown set of factors that is a developmental clock
that triggers puberty. So, these neurons are not functional before puberty
and then at some point, they become functional and release LHRH at high frequency.
The developmental clock that triggers this function is not well understood at all
but, for sure, this is one of the main controls of the function of LHRH neurons.
Now, these neurons are also sensitive to external cues,
for example, pheromonal cues or sensory stimuli that then leads to reproductive behavior.
So, it is now that pheromone detection leads to increased LHRH synthesis
and increased LHRH release.
Now, moreover, the function of these neurons is also extremely sensitive
to the internal state of the animal.
For example, an animal that is starving is not going to reproduce,
or an animal that is very stressed won't reproduce either
and this is because the other areas of the brain that deal with stress
or the level of nutrition are sending signals
that tightly control these LHRH neurons.
So, you know, the hypothalamus in general is in charge of the homeostasis of the animal
and the appropriate coordination of all the functions of the organism,
reproduction, aggression, nutrition, sleep, et cetera.
And so it's very essential, the release of LHRH has many levels of control,
both by the environment and the internal state of the animal.
So, we decided that these neurons would be a perfect target for a study
and that by investigating all the, the nature of the sensory cues that send input to those.
we will understand better the circuitry controlling reproduction and fertility in the rodent brain.
Now the second set of tools that we used are viruses
and in particular a set of viruses called pseudo rabies viruses that have the ability to replicate within neurons
and more importantly to jump across synaptically connected chains of neurons.
So, it will replicate into a neuron, then cross the synapse and reach from post-synaptic to pre-synaptic cells
and then jump again, et cetera,
and will therefore infect all the neurons that are connected to each other synaptically.
Now, the type of virus that we used
are conditional pseudo rabies viruses that have been made by Lynn Enquist
in Princeton and also Jeff Friedman at Rockefeller,
they built modified pseudo rabies virus
that when infecting the neuron, is non-functional. So, that virus, shown here in red,
infect the neuron, sorry, but is unable to replicate,
and when a neuron expresses a particular enzyme called a Cre recombinase,
that is indicated here like a pair of scissors,
it cut out a cassette of the virus genome, that makes now the virus able to replicate
and also to express the green fluorescent protein
and therefore now, the virus can replicate, jump across synapses,
and label all the infected neurons in green, fluorescent green.
And therefore, we generated a transgenic mouse line
expressing the Cre recombinase specifically in neurons expressing LHRH.
So, LHRH promoter driving the Cre recombinase
and by injecting this conditional virus in the medial pre-optic area,
the virus will infect neurons, but replicate and become green fluorescent
only in neurons expressing the neuropeptide LHRH
and therefore, one will be able to visualize very nicely all the LHRH neurons,
but also all the afferent neurons, the neurons synaptically connected to this LHRH neurons.
And so, when we performed the study, we identify a lot of infected neurons throughout the brain,
indicating that they were synaptically connected,
sending information into LHRH neurons.
And as control, we could visualize a number of brain areas
that were known to send information to LHRH neurons,
in particular, areas that provide sensory input,
that provide information about the circadian clock, suprachiasmatic nucleus
that provide information about the stress level in the brainstem
or the level of nutrition in the, from the arcuate nucleus and other areas.
So, we found all of these by infecting the, injecting the virus
by LHRH neurons, we were able to identify all these brain areas,
but our main goal was to try to investigate within the vomeronasal system
all the specific brain areas that ultimately send input to the medial preoptic area,
the neurons expressing LHRH neurons there.
And specifically, what we were hoping is that if the virus could indeed
jump from post-synaptic to presynaptic cells,
throughout enough jumping of synapses,
we could even be able to recognize specific populations of the vomeronasal organ
and maybe recognize them with the green fluorescent protein,
maybe identify what are the specific receptors that ultimately send information to LHRH neurons.
And when we performed the experiment,
when I say we, actually my graduate student Hayan Yoon,
the result was quite astonishing. Which is that we recognized many brain areas,
but within overall the olfactory and vomeronasal system,
the brain areas that we recognized in the cortical amygdala, the pyriform cortex,
the olfactory tubercule, the olfactory bulb,
and a specific population, also, very smallest population of neurons in the olfactory neurons.
Now, what's wrong here?
Well, what's wrong is that all of these belong to the main olfactory system,
and we were actually completely unable to recognize, to identify any labeled area
within the vomeronasal system. Which is that in contrast to what was found in the literature,
in which classical dye tracing experiment had identified strong connection of the medial pre-optic area
to the medial amygdala and the vomeronasal system,
what we found was instead very strong connection to the main olfactory system
and in particular, to cortical areas of the olfactory system.
So, so what's going on? Well, you know, classical dye tracing experiments
put the dye in the particular area where the LHRH neurons are located,
them among many other types of neurons,
and these tracing experiment indeed show a very strong link
between the vomeronasal system and the medial preoptic area
in which LHRH neurons are located.
Our experiment is a genetically controlled tracing experiment
in which what we visualize are the very specific connections
of these very precise population of neurons expressing the LHRH genes.
And when we do this, what we found is that it's possible that all the other neurons around
are connected to the vomeronasal system,
but the LHRH neurons are precisely not connected to the vomeronasal system
and instead are connected to the main olfactory system.
So, this is quite interesting and in particular,
it points to a particular population of neurons in the olfactory system
that send input to LHRH neurons, to neurons involved in the control of reproduction,
and therefore, these neurons are very likely to detect pheromones.
In fact, by definition,
if they are connected to areas of the brain involved in the control of reproduction,
they are pheromone detecting neurons.
We've recognize that these neurons expressing LHRH,
which is essential for the control of reproduction,
are, have this massive connection from the olfactory system.
This is obviously an anatomical finding and it's absolutely essential to have some type of functional correlates.
In other words, if indeed the main olfactory system provides such a massive input
to LHRH neurons in the medial preoptic area,
then animals that are deficient in olfactory function should also have deficiency in reproduction.
And this is indeed exactly what we found.
Remember, we found that the TRPC2 mutant male are perfectly able to mate,
but mate both with males and females.
And when we now investigated male mouse
that are deficient for the olfactory cyclic nucleotide gated channel,
so impaired in the main sense of smell,
what we found was that these animals are absolutely unable to mate.
So, they don't even recognize the female, don't even acknowledge the presence of females.
So, these provide very nice and direct functional correlate
to this massive input for the main olfactory system
into the control of reproduction.
So, in other words, it's now very clear that both vomeronasal system
and olfactory system both contribute to the perception of pheromones
that lead then to the control of reproduction and fertility in the animal.