Sex and Smell: Molecular Biology of Pheromone Perception

Part 1: Sex and Smell: Chemosensory Detection

00:00:03.28 So, today, I would like to tell you about the research done in my laboratory,
00:00:09.17 that is investigating the molecular biology of pheromone perception in mammals.
00:00:14.25 And what we are trying to do is to use molecular and genetic tools
00:00:19.18 in order to understand the basic mechanisms underlying instinctive behavior in mammals
00:00:26.07 and in the particular example of smell, the control of reproductive and aggressive behavior.
00:00:32.12 There will be three parts in my talk:
00:00:35.08 in the first part, I will introduce you to the system,
00:00:38.13 in particular the use of genes and molecular tools,
00:00:42.09 in order to investigate chemosensory detection in mammalian systems.
00:00:47.23 In a second part, I will more develop the type of research that is being performed in my lab
00:00:54.14 in order to understand the molecular mechanisms and brain circuits
00:00:58.19 that underlie pheromone perception leading to behavioral changes in the mammalian brain.
00:01:04.12 And then in the final part, I will tell you more recent results
00:01:09.24 that investigate the sex-specificity of the pheromone response.
00:01:14.13 Why do male and female of a particular species respond differently to pheromonal cues?
00:01:21.00 So, the introduction.
00:01:25.01 So, something that is very striking in animal behavior
00:01:30.21 is how in many different species, specific behaviors are really identical
00:01:37.07 among all animals of a given species, even when animals have been raised in isolation.
00:01:43.21 So, for example, within a given species of spider,
00:01:47.23 the web looks really identical,
00:01:50.15 or the nest building of birds is also very striking,
00:01:54.21 a given species of bird will build a very sophisticated nest with very specific morphology
00:02:02.28 over and over again, even though animals are raised in isolation
00:02:07.25 and really don't have the ability to learn from the other animals of the species.
00:02:13.08 Similarly, regarding social behavior, courtship behavior, maternal behavior,
00:02:20.03 social behavior to the large extent also in some species appear very reproducible
00:02:26.22 among all the animals of the given species.
00:02:30.06 And these really raise a very specific question, which is, what are the mechanisms
00:02:37.07 that really control those behaviors, what is the relationship between genetic information
00:02:44.08 that really provides the animal with this information that are common to all the species
00:02:54.11 and the function of the brain?
00:02:56.05 And we are particularly interested in studying this type of issue
00:03:01.21 in mammals and in, particularly, in rodents.
00:03:04.25 And the type of question that we are raising are as follows:
00:03:10.19 first, we, it's very clear that a number of behaviors within the animals
00:03:17.18 result from the animal investigation of the environment
00:03:22.20 and the response to the environment will lead in turn
00:03:27.01 to a number of brain functions that will then lead to specific behaviors.
00:03:34.17 Now, it's very important to understand that the brain is not sensitive directly
00:03:41.11 to the environment; the environment provides information about temperature,
00:03:47.08 about the level of light, about the presence of pressure waves,
00:03:53.22 that forms sounds, or the presence of chemicals.
00:03:57.09 The brain is not sensitive to these physical and chemical properties of the environment,
00:04:03.13 the brain is sensitive to just neural impulses
00:04:07.16 and so animals have developed specialized organs, or sensory organs,
00:04:13.12 that are able to translate the specific information that is present in the environment,
00:04:20.00 for example, mechanical signal, or temperature, the presence of photons,
00:04:25.09 the presence of chemicals in the environment,
00:04:29.16 these sensory organs translate those chemical and physical properties
00:04:34.08 into electrical signal, neuronal signal,
00:04:37.22 and these neuronal signals are then encoded within the brain
00:04:41.10 and leads to very specific perception and behavioral responses.
00:04:46.23 Now, these seem very obvious, but it is not that obvious,
00:04:51.17 and I think to illustrate how things could go wrong,
00:04:55.21 I would like to describe to you a very interesting condition
00:04:59.12 happening in human in which these phenomenon, these mechanisms,
00:05:05.05 are actually going wrong.
00:05:07.24 And this is a syndrome called synesthesia,
00:05:12.24 in which people experience the blending of two or more senses.
00:05:18.00 And to illustrate this is this very nice image taken from an article in Scientific American
00:05:25.12 in which the, that illustrates some of the anomalies in the perception of people with synesthesia.
00:05:34.24 So, for example, when they are hearing a sound,
00:05:39.24 it brings the perception of a color in our brain.
00:05:43.26 Or when they are tasting a particular food, a shape, the perception of a shape
00:05:51.07 comes into their brain. So, you might wonder what's going on here?
00:05:55.19 And is there a possibility that people who associate so tightly
00:06:01.00 a number or a letter with a particular color,
00:06:04.06 after all, might just be, this might just be resulting from a particular experience.
00:06:12.10 In childhood, for example, they had magnet on their fridge that had number with specific colors
00:06:18.13 and therefore, that's how they see.
00:06:20.28 And, some scientists were able to demonstrate very clearly
00:06:26.21 that is was not coming from sensory experience, from previous experience,
00:06:32.01 or training, but rather from some very unusual properties of the brain,
00:06:38.10 the function of the brain, of these persons.
00:06:41.27 And what illustrates this extremely well is the following test,
00:06:47.28 in which what you can see here is a collection of numbers
00:06:51.23 and you see if you look closely, it's mainly 5,
00:06:55.26 if you look a little bit more closely, there are some number 2 around.
00:07:00.25 If you were looking very closely, it would take you a few minutes
00:07:04.15 to find where all the number 2's around, intermingled within the number 5.
00:07:10.12 But a person affected with synesthesia would actually tell you right away
00:07:16.28 where the number 2's are because the people who have synesthesia
00:07:21.27 and see numbers with specific colors would immediately have the number 2
00:07:26.09 striking out as having those very specific colors.
00:07:30.24 So, in two seconds, they would immediately tell you where the number 2's are.
00:07:34.15 So, this is just to give you an example of the difference between detecting
00:07:40.03 a particular sensory stimulus, and the perception of the stimulus.
00:07:44.20 We take it for granted that a pressure wave leads to a particular sound,
00:07:51.03 or a particular wavelength of photons gives rise to particular color,
00:07:56.23 but in fact, the brain is the one that establish the link
00:08:01.11 between a particular sensory detection and the associated perception.
00:08:06.07 And this goes through a number of layers within the brain,
00:08:10.00 and what my lab is interested in is understanding what these layers are,
00:08:13.24 what are the mechanisms that lead from the detection
00:08:16.12 to the perception to the change in behavior.
00:08:19.00 Now, our system of interest is the olfactory system
00:08:24.03 and the primary event in olfactory detection is happening at the level of olfactory epithelium,
00:08:32.08 in the nasal cavity, in which specific specialized neurons called olfactory neurons
00:08:37.26 are located. These neurons are bipolar neurons, they have a cell body in the olfactory epithelium,
00:08:45.03 a sensory dendrite that terminates with a sensory structure called an olfactory knob,
00:08:52.03 and this is the structure in which the receptors and the ion channel are located,
00:08:56.03 that would lead to the chemical signal transduction
00:08:59.20 that will then be translated into an electrical signal
00:09:02.29 and the second, the axonal portion of this neuron then projects to the brain.
00:09:07.28 Now, there are different types of olfactory signal.
00:09:14.06 The olfactory signal we are mainly familiar with
00:09:17.16 are signals that provide some information about the environment,
00:09:22.10 the presence of food, the presence of enjoyable stimuli,
00:09:27.27 the presence of unpleasant stimuli or maybe a danger,
00:09:32.17 something to avoid, and the primary event happened at the level
00:09:37.11 of the olfactory cavity where specific receptor molecules
00:09:42.22 detect particular odorants and this information is then translated
00:09:47.28 and transmitted to the brain and leads to behavioral responses.
00:09:52.16 Now, these types of responses to food or pleasant or unpleasant stimuli
00:09:59.19 are to large part result from your experience throughout life,
00:10:06.02 and things that you've been enjoying, or enjoying less,
00:10:10.03 and so the cortical areas of the brain
00:10:14.06 are thought to be responsible from this type of processing
00:10:18.22 that, again, can be largely modified by experience.
00:10:23.21 So, for example, you can have a very unpleasant encounter associated with a particular perfume,
00:10:30.05 and you will hate this perfume all throughout your life.
00:10:33.25 Another person will have a very different reaction to that particular smell.
00:10:39.02 Now, another type of chemical signals that are perceived by animals
00:10:44.02 are the pheromones.
00:10:45.14 The definition of pheromones are chemicals that are emitted
00:10:49.07 by animals within a species and enable these animals to communicate with each other.
00:10:54.18 For example, providing information about the gender of the animal,
00:10:58.16 this is a male or female, or is it an animal that is a kin,
00:11:04.09 so it provides information about the identity, the sexual and genetic identity of the animal.
00:11:11.14 It enables them to establish the animal group,
00:11:16.27 recognize dominant animals, recognize animals that are ready to mate,
00:11:21.27 recognize rivals, et cetera.
00:11:23.28 What is very striking about pheromones is this leads to
00:11:29.14 what are thought to be instinctive behaviors,
00:11:32.17 that lead to aggressive behavior or mating behavior.
00:11:36.17 So, those are really chemicals that are essential
00:11:39.15 for the reproduction and the survival of the species
00:11:42.17 and obviously, an animal cannot re-discover at each new generation
00:11:49.06 who are the potential mate or the potential rivals
00:11:54.13 and so these types of circuitry are thought to be largely innate
00:12:00.19 and lead to the trigger of these very reproducible behavior
00:12:07.01 within an animal species.
00:12:08.29 And this is particularly striking in rodents that are mainly recognizing their environment
00:12:17.07 using olfactory cues and therefore provide a terrific experimental system
00:12:23.09 in order to study the molecular biology of smell,
00:12:27.27 the molecular biology of pheromone detection,
00:12:30.07 and how are behaviors initiated from the stimulation of these sensory signals.
00:12:35.08 So, this is a cartoon that summarizes very briefly
00:12:39.23 what has been the work that has been done,
00:12:42.14 mainly in the laboratory of Richard Axel and Linda Buck,
00:12:46.05 on the molecular biology of smell.
00:12:49.04 So, odorants are detected in the nasal cavity by a structure called the olfactory epithelium,
00:12:56.01 and neurons in the olfactory epithelium, here represented by these different colors,
00:13:02.19 each express a different olfactory receptor gene,
00:13:06.11 and therefore, each population of olfactory neuron
00:13:09.29 can recognize a very specific subset of odorant signals.
00:13:14.25 These odorant signals are detected, lead to the excitation of these olfactory neurons
00:13:20.18 that will transmit information to the brain,
00:13:22.28 and in the brain, they form specific structural glomeruli
00:13:26.22 that are essential for decoding of olfactory information
00:13:31.29 in the first relay station of the brain called the olfactory bulb.
00:13:36.06 So, this site is thought to be mainly responsible from the detection of odorant signals
00:13:44.18 and lead to the cognitive perception of a smell.
00:13:48.06 And in contrast, it has been assumed for a long time,
00:13:53.10 and I will discuss this much further in detail during this presentation,
00:13:58.22 the detection of pheromones is thought to occur in a different structure of the nose,
00:14:03.26 called the vomeronasal organ, or VNO for short,
00:14:07.21 and this structure is very different morphologically.
00:14:11.14 It's in the septum of the nose,
00:14:13.22 so the septum is the cartilage that is in the middle of the nose,
00:14:16.24 it has a tubular structure, such that the pheromones
00:14:21.06 come into contact with the vomeronasal neurons that are located here
00:14:28.00 through a lumen. The lumen is filled up with liquid,
00:14:31.20 and therefore, the pheromones are mainly thought to be non-volatile molecules
00:14:37.05 that are pumped out of the nasal cavity by the vomeronasal, the VNO structure,
00:14:44.03 and the neurons from the VNO express two types of receptors,
00:14:49.01 called V1R, a family of pheromone receptor that we identified many years ago,
00:14:55.08 and the V2Rs, a distinct family of receptors,
00:14:59.11 and neurons expressing these two distinct receptor families
00:15:03.12 then project to an area of the brain called the accessory olfactory bulb,
00:15:08.10 and in a manner slightly different than what's happening in the main olfactory bulb
00:15:13.12 that we've seen before, these chemicals, the signal triggered by the chemical
00:15:21.04 is then processed by this area of the brain
00:15:24.04 and then sent to higher brain structures.

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.

Part 3: Sex-Specificity of Pheromone Responses

00:00:04.15 So, in the next slide, in the next part of my talk, I will then address a different point
00:00:11.28 which is the sex specificity of the pheromone response.
00:00:14.28 We've talked about the control of reproduction and reproductive behavior in the males,
00:00:22.00 but what's happening in the females?
00:00:24.19 And how do animals know what to do according to whether they are males or females.
00:00:29.21 And so, what we've seen in the previous part
00:00:33.25 of this talk is that the sense of smell, or actually it's a portion of the sense of smell,
00:00:39.13 detects chemicals that are essential for the control of mating behavior
00:00:44.19 and provide direct information to neurons expressing the key modulator of reproduction
00:00:52.01 and fertility, which is LHRH.
00:00:54.00 And the vomeronasal system, as demonstrated by the phenotype of the TRPC2 mutant,
00:01:00.04 provides gender identification and therefore, these two systems are working hand by hands.
00:01:07.13 But this, in fact, is what's happening in the male mouse,
00:01:11.17 and Tali Kimchi, a postdoctoral fellow in my lab,
00:01:16.05 asked the question about female behavior
00:01:19.29 and in particular, how could male and female trigger different behavior
00:01:25.23 when detecting a similar pheromone, such that a male detecting another male
00:01:31.23 will attack that male, but a female detecting a male will trigger mating behavior instead.
00:01:41.16 So, Tali, in the lab, just performed a very simple behavior experiment
00:01:50.10 which is she observed the receptivity of the mutant females.
00:01:56.11 So, the female mutant is here in grey
00:02:00.04 and the male now is here in black and what she wanted to observe
00:02:04.22 is how receptive is the mutant female to the male approaching her.
00:02:12.21 And as you can see, in the video, the female is really behaving very strangely.
00:02:26.16 In fact, not behaving at all like a female.
00:02:30.00 But, rather displaying typical sexual behavior of a male.
00:02:34.20 So, that was absolutely astonishing
00:02:37.15 and I actually went and watched the animal and made sure it indeed a female,
00:02:43.04 it is indeed a female.
00:02:45.11 And when we carefully looked at the behavior
00:02:49.17 of the mutant female, so what you have here in pink is the number of animals
00:02:55.10 presenting mounting behavior towards other females and towards males,
00:03:00.21 in pink are the mutant female, red are the wild-type female,
00:03:04.24 blue are the wild-type male, light blue mutant male, TRPC2 mutant male,
00:03:09.16 and what is very striking is that the mutant female behaves exactly like the male
00:03:16.00 towards a female intruder, and also, is also trying to mate as a male
00:03:22.20 with male intruders.
00:03:25.27 And these are more characteristic of male sexual behavior,
00:03:30.02 in which you can see again that the female mutant displays male-typical sexual behavior,
00:03:36.14 both towards females and towards male intruders.
00:03:41.00 So, that was very, very striking. Why is the female mutant behaving like a male?
00:03:47.24 We've seen that the male mutant is unable to discriminate the sex of conspecifics,
00:03:54.06 so will mate both towards males, with males and female conspecifics.
00:03:59.19 But here, it seems that the female is not confused about the gender of the other animals,
00:04:04.28 but confused about her own sex identity.
00:04:07.24 And so that was very, very surprising.
00:04:11.08 And we looked at many signs of male-typical sexual behavior
00:04:19.03 and we found that the female mutant displays all characteristics of male sexual behavior,
00:04:26.16 the female mutant.
00:04:27.23 We then looked at other types of behavior that are grouped under the term
00:04:33.06 "courtship behavior", in particular, what we call solicitation behavior,
00:04:37.10 which is the male, normal male, puts its snout under the body of the female
00:04:43.16 and raise the body of the female.
00:04:45.10 So, this, again, is a behavior that is also highly displayed by the mutant female,
00:04:52.00 and never displayed by the wild-type female.
00:04:56.08 And again, it's displayed both towards female intruders and also towards male intruders.
00:05:02.09 So, the female mutant both is confused about her own gender identity
00:05:07.16 and behaves like a male, but also exactly like the TRPC2 mutant,
00:05:11.16 also seems to have exactly the same behavior towards both males and females,
00:05:15.22 so it's also unable to discriminate the gender identity of the other animals.
00:05:20.13 Another behavior, courtship behavior that we looked at
00:05:26.13 is the emission of ultrasound vocalization.
00:05:29.03 So, male mice, normal male mice, emit ultrasonic whistle when they detect a female.
00:05:41.14 And, again, we found that the mutant female emits ultrasound exactly like the males,
00:05:48.28 and emit them both towards males and female intruders.
00:05:52.18 Now, what is interesting about ultrasound vocalization
00:05:55.14 is that you can look not only at them quantitatively, but also qualitatively,
00:06:00.07 which is that if you, this is the sonograms,
00:06:05.00 so the ultrasounds are these high-frequency pitch,
00:06:07.29 emitted by males in response to females,
00:06:12.01 and you can see these complex sonograms here that show pitch jump
00:06:16.20 and very complex sounds, ultrasounds, that the wild-type female never emits,
00:06:24.03 so there's almost no ultrasounds, and whatever ultrasounds the female emits
00:06:29.06 are kind of very simple in nature.
00:06:31.24 And what we found is that the mutant female not only emit a lot of ultrasonic vocalization,
00:06:38.00 but also those are quite complex, like the males.
00:06:41.20 So, again, for all the types of behaviors that we observed,
00:06:46.28 whether those are purely sexual behaviors, or part of what I call courtship behavior,
00:06:52.08 the female really seem to behave like a male.
00:06:55.12 And again, you know, this is very surprising.
00:06:57.28 So, what's going on?
00:06:59.01 Well, there are two possible explanations of this behavior.
00:07:03.11 One is we are observing a mutant animal in which the vomeronasal organ
00:07:08.17 has never been functional.
00:07:10.14 And because it has never been functional,
00:07:13.02 and because there's a lot of experiments done in many sensory systems,
00:07:18.01 that indicate that the function of, for example, the visual system or the auditory system,
00:07:24.00 is very important early in development,
00:07:26.18 it's possible that an animal that has never experienced any VNO function
00:07:31.17 has a lot of miswiring in the brain, and therefore the female behaves as a male
00:07:37.01 because of this miswiring.
00:07:39.07 Alternatively, the brain of these animals, these mutant animals,
00:07:44.02 might be perfectly fine, but the phenotype of the mutant
00:07:48.29 just indicates that the vomeronasal organ
00:07:51.09 is important to constantly repress male behavior by females.
00:07:56.21 And so, in order to address this question, we performed a very simple experiment
00:08:03.16 which is to remove, surgically, the vomeronasal organ
00:08:07.19 from an adult female.
00:08:09.26 So, you can just perform the surgery through the palette,
00:08:13.13 grab the vomeronasal organ, remove it,
00:08:16.10 wait for a few weeks until the nasal cavity heals,
00:08:20.19 and the nasal cavity becomes cleared,
00:08:23.15 and we perform behavioral experiments on those animals
00:08:27.23 and after the behavioral experiment, sacrifice the animal
00:08:33.06 and confirm that there were no vomeronasal organ anymore,
00:08:37.07 but that the nasal cavity was perfectly cleared,
00:08:40.01 and by looking at the brain of these animals, we can also confirm that
00:08:44.11 there are no axonal projections to the accessory olfactory bulb.
00:08:47.27 So, there's really no VNO neurons anymore.
00:08:49.28 So, in this animal in which the VNO has been removed,
00:08:54.02 this VNOx, what's happening?
00:08:55.16 Well, this is shown in this boxed part of the histogram,
00:09:04.13 as you can see, here for number of animal mounting,
00:09:08.28 or the number of, the percentage of sexual behavior,
00:09:13.25 these animals without a functional vomeronasal organ behave exactly like the males.
00:09:19.06 And so, and exactly like the mutant female.
00:09:23.03 In other words, the behavior of the male-specific behavior of the mutant female
00:09:29.10 is not, does not originate from a mis-wired brain, but instead comes from
00:09:35.28 the absence of the repression of the male behavior
00:09:39.29 by the female.
00:09:41.18 Now, when Tali Kimchi, the postdoc in the laboratory who was doing these experiments,
00:09:49.16 she also realized something very important to understand a little bit the history
00:09:56.19 of the field.
00:09:57.13 So, when Tali performed the surgical ablation of the vomeronasal organ,
00:10:02.18 she realized that when she was performing the experiment,
00:10:06.13 as was described in the literature, in all these classical surgical ablation of the VNO
00:10:12.27 she was not obtaining good results.
00:10:15.18 And she observed that the behavior of these animals was kind of,
00:10:19.11 seemed to be, suboptimal.
00:10:21.06 And in fact, when she sectioned and observed the nasal cavity of these animals,
00:10:28.28 what she found was that the nasal cavity was entirely, were entirely obstructed by blood clots.
00:10:35.01 And therefore, it was very likely that both their vomeronasal organ was gone
00:10:40.20 but also that their olfactory function was impaired
00:10:44.03 because the nasal cavity was just entirely obstructed.
00:10:47.05 And so she modified the protocol, let the animal recover slightly longer,
00:10:52.17 and also aspirated these blood clots on a daily basis in order for the animal to have a very clear nasal cavity
00:11:01.18 and then no vomeronasal function.
00:11:04.02 So, it is quite possible at this point that the discrepancy that we have obtained
00:11:10.08 between the result obtained with, the discrepancy between the result obtained
00:11:15.28 by surgical ablation, that showed defects in mating behavior and aggressive behavior
00:11:21.07 and our results with the TRPC2 mutant
00:11:24.06 might be due to the confusion of really the real sensory defect on these animals with classical surgical ablation,
00:11:33.14 in which those animals were both, might have been both partially at least, olfactory impaired,
00:11:40.09 and therefore showed abnormal mating behavior, together with the lack of aggressive behavior.
00:11:47.02 Now, what is very interesting is that the female, without a vomeronasal organ,
00:11:55.03 when the vomeronasal organ was removed,
00:11:57.18 in the adult females, also start to emit ultrasound vocalizations,
00:12:05.09 exactly like the mutant female and this is quite remarkable
00:12:09.29 because people had attributed the lack of ultrasound emitted by the females
00:12:15.09 by possibly the lack of vocal organs to emit these ultrasounds.
00:12:20.03 So, I think it's quite remarkable that after few weeks after VNO removal
00:12:24.09 these females are perfectly able to emit complex ultrasonic vocalization.
00:12:30.25 So, we've shown, therefore, that these mutant females display male-like traits.
00:12:38.10 And at this point, we were still worried about one particular caveat,
00:12:42.07 which is that, as I mentioned earlier, any type of social behavior,
00:12:47.03 reproductive behavior, aggressive behavior,
00:12:49.12 is really highly influenced by the environment of the animal,
00:12:53.25 so, we also observed the behavior of these mutant females in large arena
00:12:59.29 in which the animals are kept in these 1.2 by 1.2 meter arenas
00:13:07.15 for several weeks at a time in a very enriched environment
00:13:11.05 with little shelters and complex environment
00:13:13.21 and video recorded for a long period of time.
00:13:16.28 And then we used a software to really analyze in very much detail
00:13:23.27 every single behavioral attempt by the animal.
00:13:28.02 And so this video shows again the behavior of the female mutant
00:13:35.19 in the presence of males.
00:13:37.19 And this is a clip that has been done when the males have just been introduced
00:13:41.13 to the cage and what you can see
00:13:46.15 is that exactly as was found in the small cages, the females are literally mobbing the male
00:13:53.05 and attempting to show this male-like display with the wild-type animals.
00:14:00.22 So, in other words, what we have observed in small cages
00:14:05.11 seems to hold true also in these large arenas.
00:14:08.29 But, what's happening is that when we keep these animals in the arena for a long time,
00:14:14.01 so the wild-type male and the mutant female, the male becomes more and more aggressive
00:14:18.16 and ultimately mates successfully with the females
00:14:21.23 and so all the mutant females end up being pregnant.
00:14:25.05 And so that's actually, then enabled to perform another set of observations,
00:14:30.21 which is the maternal behavior of the mutant female compared to the heterozygous female.
00:14:38.02 And what we found was quite interesting,
00:14:40.19 which is when we looked at the amount of time the female spent in the nest,
00:14:46.16 mice have communal nests, in which all the pups are put, and then the female just takes turn
00:14:52.01 around the nest, we found that, as expected, the non-mutant females
00:14:58.24 spend a lot of time in the nest,
00:15:00.29 but the mutant female is spending less and less time,
00:15:04.23 and in fact at some point, even, we removed the divider between the mutant colony
00:15:10.21 and the non-mutant colony and the mutant female
00:15:13.16 immediately abandoned the nest to explore the new territory.
00:15:17.08 So, this mutant female therefore have a very clear defect
00:15:21.08 in maternal behavior. So, that's very interesting.
00:15:24.19 To summarize, the mutant females show male-like behavior,
00:15:28.25 and show a defect in female specific behavior.
00:15:32.17 Now, all these types of behaviors
00:15:35.01 are known to be under the influence of steroid hormones
00:15:39.14 and we therefore wondered whether the level of steroids were different in the mutant
00:15:44.14 rather than wild-type animal.
00:15:48.17 And so, when we checked the level of testosterone, or the level of estrogen,
00:15:53.21 in the animal, we actually found that there was no difference
00:15:57.18 between wild-type animals and mutants, which is that both for males and females,
00:16:05.27 female mutant had female specific levels of estrogen
00:16:11.01 and didn't have elevated level of testosterone,
00:16:14.22 and therefore, one could not attribute the male typical behavior of the mutant female
00:16:21.21 to abnormal level of testosterone.
00:16:24.26 So, but again, this is quite surprising because it entirely challenges
00:16:30.03 the ideas that we have now regarding the male and the female brains.
00:16:36.23 So, the assumption regarding the difference in behavior between males and females
00:16:43.26 are as follows:
00:16:46.01 so, it is thought that steroid hormones have two roles in establishing the male brain.
00:16:55.06 One is that steroids are elevated, and testosterone in particular,
00:17:00.15 around birth and this has an organizational effect
00:17:05.08 such that specific set of connections and nuclei can be formed
00:17:10.04 in the developing male brain,
00:17:12.26 and that in the female, the absence of this surge in testosterone
00:17:18.07 leads to a different type of differentiation
00:17:21.11 which is typical of females.
00:17:23.18 And at puberty, a second surge of testosterone leads to the activation of this male-specific circuit,
00:17:31.19 whereas, in the female, female steroids lead to the activation of female specific circuitry.
00:17:37.22 But what we found is that in fact, the male, adult female,
00:17:46.29 without a functional vomeronasal organ,
00:17:49.11 whether this is come from a genetic mutation or the surgical ablation,
00:17:53.05 is perfectly able to display male-specific behavior.
00:17:58.09 And therefore, we have to draw this circuitry quite differently
00:18:03.05 and assume that in the female brain are both functional, potentially functional, male circuit
00:18:11.16 and female circuit and those are under the control of a switch,
00:18:16.27 if you wish, that is controlled by the vomeronasal organ
00:18:22.19 and therefore, the vomeronasal organ enables the female to have a female-specific behavior,
00:18:30.24 and represses a male-specific circuitry
00:18:35.08 that does exist and is potentially functional in the female brain.
00:18:39.20 And if one removes the switch, the vomeronasal organ,
00:18:44.23 either genetically or surgically,
00:18:47.11 then one can observe these potentially functional male circuit.
00:18:54.04 Now, there's a lot attraction to this model;
00:18:59.06 one of them is that although people had postulated
00:19:03.05 that male and female brain had very different circuits, and nuclei,
00:19:08.17 that would explain the difference in behavior between males and females,
00:19:13.25 well, it just happened that people had serious trouble to identify circuits,
00:19:19.09 and nuclei, that were distinct between males and females.
00:19:23.04 People have looked for decades to the anatomical correlates in the brain
00:19:28.22 of male and female specific behaviors
00:19:33.00 and all what was found was slight differences either in the size of specific nuclei
00:19:40.22 or the composition in neurotransmitters,
00:19:42.26 and other types of small, quantitative differences,
00:19:47.27 when the observation of male and female specific behaviors
00:19:51.28 shows that differences must be qualitative differences
00:19:55.25 and not only small, quantitative differences.
00:19:58.11 So, in contrast, a model like this, in which both males and female circuit exist in the brain
00:20:06.07 and are under the control of a switch, a sensory switch,
00:20:10.18 that is itself sexually dimorphic, that is a very attractive model.
00:20:16.09 Now, obviously, for sake of balance, one has to postulate
00:20:21.02 that in the male brain, there is also both a male and a female circuit
00:20:26.03 that are both potentially functional and are under the control of the vomeronasal system.
00:20:33.03 So, is this reasonable now?
00:20:37.12 Well, very interestingly, some old literature shows that if one removes the vomeronasal organ
00:20:46.03 from an adult male, or impairs specific nuclei of the vomeronasal system,
00:20:51.04 then male rats appear to suddenly display maternal-like behavior,
00:20:59.00 retrieve the pups, bring the pups to the nest, build the nest,
00:21:03.26 and have a pseudo nursing behavior that is never observed in the wild-type rats.
00:21:10.23 So, therefore, the removal of the vomeronasal organ seem indeed
00:21:16.03 to let female specific behavior emerge in the male
00:21:21.08 and therefore, for all intent and purposes, I'm going to suppose and I'm going to postulate
00:21:28.08 that the male, like the female brain, have both male and female circuit
00:21:32.20 that are potentially functional
00:21:34.00 and themselves under the control of vomeronasal system,
00:21:39.12 vomeronasal function. Now, this is a very attractive model,
00:21:44.11 also, makes a lot of sense from a developmental standpoint
00:21:48.05 because an entire circuit that is built differently in the male and in the female
00:21:55.28 is quite difficult to conceive, it's difficult to build,
00:22:01.19 an entirely different set of connections in the female brain and in the male brain.
00:22:06.08 This, in contrast, is, seems to be much easier. There is one brain
00:22:12.03 that is built under a very complex set of genetic information
00:22:16.13 and then there is a switch, the repression of the wrong behavior
00:22:21.02 and the activation of the right behavior
00:22:23.00 that is performed by one specific system, which is the vomeronasal cues.
00:22:30.14 Now, this model has quite a number of implications from an evolutionary standpoint
00:22:37.10 and I would like to finish this presentation
00:22:41.12 by what I think are a number of really interesting speculations
00:22:46.05 of what the role of a system like this could be during evolution.
00:22:52.17 One of them is this system really provides quite a lot of flexibility
00:23:01.07 to evolve certain behavioral traits in animals that are very close genetically.
00:23:08.26 And all you need is to have an animal that, for example, in female,
00:23:16.07 will take one part of the circuit that is usually expressed by the males
00:23:21.26 and then suddenly activate that circuit, and vice versa for the males.
00:23:26.15 So, in other words, you would have some type of inversion of some of these arrows here
00:23:32.25 such that, for example, a male could, in a species very close to another one,
00:23:38.24 suddenly express a trait that is seen more as a female trait.
00:23:42.29 And this does exist in nature and has been described,
00:23:47.06 in particular in this very interesting animal called the vole,
00:23:51.09 there are two type of vole, two species of vole that are quite close genetically
00:23:57.03 that have been described, the mountain vole and the prairie vole.
00:24:00.22 Now, the mountain vole, the males are promiscuous, they mate with a female and then abandon the nest.
00:24:08.14 The prairie vole in contrast, the male of the prairie vole has a very different behavior,
00:24:14.11 the couple are totally monogamous and the dad is taking care of the pups
00:24:21.00 and have a lot of parental traits that are seen in the female
00:24:27.24 in the other species. And so one can see that this could be some type
00:24:32.13 of part of the maternal-like circuit that has actually been adopted as a male-like behavior
00:24:42.04 in the prairie vole, compared to the mountain vole.
00:24:45.24 And you know, we can imagine, like, these animals that are very similar to each other
00:24:51.26 genetically, could evolve specific traits
00:24:56.03 that are thought to be classically displayed by the other sex.
00:25:01.09 We know, and this type of behavior, or evolution,
00:25:07.04 would be extremely hard to explain with the classical model
00:25:11.16 of male and female brain being so distinct.
00:25:15.27 We know some of these examples,
00:25:18.02 the old dads and the new dads, and the new dads.
00:25:21.17 These are obviously a new generation of human behavior
00:25:26.12 that behave, new generation of humans that behave very differently
00:25:33.00 than their dad or granddad,
00:25:35.11 which assumed that taking care of their children would be absolutely non-male-like
00:25:44.07 and this is obviously complete speculation,
00:25:47.10 and more presented here as a joke,
00:25:49.29 but it's very clear that one can in the course even one or two generations,
00:25:55.18 have a very different type of behavior
00:26:01.05 and this obviously could very well come from a lot of flexibility that is offered by these models.
00:26:09.27 Now, what we've seen are very small changes in behavior,
00:26:15.16 so, somewhere in part of this circuitry.
00:26:19.21 Something more profound could happen and this would be
00:26:24.00 an entire change of the entire repertoire of behavior.
00:26:27.28 In other words, a female now behaves exactly like a male,
00:26:32.11 or a male exactly like a female.
00:26:34.04 And it's interesting to look throughout evolution
00:26:37.12 that there are indeed animal species that do that, constantly.
00:26:41.23 So, there are specific species of fish that, in which, the female switches into male
00:26:49.04 and then from male comes back to a female behavior.
00:26:52.04 And again, this would be extremely difficult to explain
00:26:56.21 if one considers that the male and the female brain have very distinct behavioral circuits;
00:27:02.25 it's much easier to explain if one considers that the brain in each species
00:27:08.22 is essentially bisexual and there's just a switch that needs to be modified.
00:27:13.08 So, I mentioned that again, in the fish, there are a certain number of fish
00:27:18.06 that can switch their behavior from males to females
00:27:21.15 sometime in a matter of days, sometime in a matter of hours,
00:27:26.28 sometimes even in a matter of minutes.
00:27:30.26 And so, again, I think this scheme provides a very interesting template
00:27:36.29 to explain these type of behaviors.
00:27:39.18 A little bit further in evolution, a very fascinating model
00:27:44.00 is the model of the whiptail lizard,
00:27:46.25 in which this particular species of lizard has only females
00:27:53.13 that reproduce by cloning,
00:27:57.01 so in sort of pathogenetic manner and what is interesting
00:28:00.28 is that the females within these species adopt a female and then a male-like behavior
00:28:08.19 although there are only females within the species
00:28:11.12 and these depend on the specific level, the ratio of progesterone and estrogen
00:28:17.17 and the, one of the females, will adopt a male-like behavior
00:28:22.17 that will help the other female to ovulate and then in turn the animal that has a male-like behavior
00:28:30.13 now will adopt the female behavior, et cetera.
00:28:33.04 Now, what is quite interesting is that they still, the ancestral species
00:28:39.21 in which male and female still existed,
00:28:43.20 and this is the case here, you have a closely related species
00:28:49.04 from which the pathogenetic one evolved
00:28:52.14 and in here you have a male and a female that show a specific male-female sexual behavior
00:29:00.23 and the two females of the pathogenetic species
00:29:04.08 do behave and show exactly the same traits,
00:29:08.08 showing that these females have kept both the male and female behavior circuit
00:29:14.00 and are just able to switch from one behavior to the other
00:29:17.18 and this fascinating work has been done by David Crews in Austin, Texas,
00:29:23.21 and his group and this is an absolutely fascinating model.
00:29:27.03 So, just to conclude this talk, and to talk about our future plan,
00:29:33.28 I think that this new model of the male and the female brain
00:29:38.05 that extend from the fish or the reptiles to mammals
00:29:43.18 is now opening really new avenues of research
00:29:48.04 on the control of male and female specific behavior.
00:29:51.29 What we are interested in doing in rodents is to look at this model
00:30:00.12 in a more mechanistic way, which is what specifically are the switches,
00:30:05.05 what are the brain areas that function as switch, how do they function,
00:30:08.28 this sexually dimorphic area here, that controls the downstream male and female circuitry,
00:30:16.04 what are those circuit components, how do they operate,
00:30:20.04 and can we have a switch that operates in a matter of months,
00:30:24.16 weeks, days, hours, minutes, these are all mechanistic questions
00:30:29.14 that we would like to address.
00:30:30.16 And thank you very much.

Videos in this Talk

Part 1: Sex and Smell: Chemosensory Detection
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: Molecular Biology of Pheromone Perception
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 3: Sex-Specificity of Pheromone Responses
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad

Speaker: Catherine Dulac

Total Duration: 1:29:52

Recorded: December 2007

All Talks in Neuroscience

Talk Overview

Pheromones have evolved to signal the sex and the dominance status of animals and to promote social and mating rituals. In this lecture, I discuss the how pheromone sensing operates in mammals. I will discuss the molecular biology of the chemosensory receptors that are involved the first steps of pheromone sensing. At a higher level of complexity, I will discuss a distinct olfactory structure called the vomeronasal organ (VNO) and how it contributes to sex-specific behavioral responses.

Speaker Bio

Catherine Dulac

Catherine Dulac is an Investigator with the Howard Hughes Medical Institute and Professor of Molecular and Cellular Biology at Harvard University. She received her Ph.D. degree from the University of Paris and worked as a postdoctoral fellow with Richard Axel at Columbia University. Dr. Dulac identified the first candidate genes encoding mammalian pheromone receptors. Dr…. Continue Reading

Part 1: Sex and Smell: Chemosensory Detection

Part 2: Molecular Biology of Pheromone Perception

Part 3: Sex-Specificity of Pheromone Responses

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Catherine Dulac

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Sex and Smell: Molecular Biology of Pheromone Perception

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