Unicamp scientists took an important step towards understanding how the exchange of signals between animals acts to trigger behaviors, emotions and influence interactions between individuals, with possible implications for the human species.

In an article published in the international journal BMC Biology, doctoral student Thiago Nakahara and his advisor, professor Fabio Papes, from the Department of Genetics and Evolution at the Institute of Biology (IB), describe the discovery of a population of neurons in the nose of mice associated with infanticide – the massacre of puppies by adult males who have not yet started their sexual life.

“In mice, virgin males, who have not yet had sexual intercourse with the female, are aggressive against puppies, which, of course, are not theirs, they belong to other males,” explained Papes. In a population of these animals, 70% of virgin males kill other people's offspring, as detailed by the professor. “It is a behavior that depends on the exchange of chemical signals, odors that are released by the puppies and (...) detected by the virgin male”.

 The biological function of infanticide is very clear, said the researcher: “The virgin male eliminates other people's offspring, those containing genes from other males.” He further explains that this is an adaptive behavior, which fulfills an evolutionary function, as it eliminates offspring from other males and increases the chance of the perpetrator transmitting his own genes to the next generation.

Sex, age and RNA

The article in BMC Biology, which also has as co-authors researchers based in the United Kingdom and the United States, draws attention to the fact that the sensory cells discovered at Unicamp are the first ever identified with a specific molecular relationship with the detection of puppies. Furthermore, they are the first olfactory neurons found to have differentiated activity according to the sex and social status of the animal, since the violent reaction triggered does not occur in females, nor in males that have already started their sexual life.

“The behavior we study is instinctive, generated in the animal without any prior exposure. The animals used in the study, virgin males, had not previously had contact with puppies. These are behaviors that appear without learning and without prior memory,” said Papes.

The neurons were discovered inside an organ present in the mouse's nasal cavity, called the vomeronasal organ. “We started this study using a modern approach, RNA sequencing, to find which genes are expressed in this sensory organ”, reported the researcher.

All cells in an animal's body have the same DNA: it is the way in which genes are expressed that differentiates, for example, skin cells from those of an internal organ. And the immediate product of DNA expression are RNA molecules, which will later control the production of proteins.

“The neurons we studied have a specific functional characteristic: they are olfactory sensory neurons, which detect odors. In these neurons, of all the genes that exist in the mouse genome, only a few are functioning, and we can find out which ones they are by evaluating the RNA”, described Papes. After sequencing the RNAs, the team had access to all the genes expressed in the olfactory organs, and that is how they discovered some sensory neurons characterized by the expression of a previously unknown odor receptor gene.

Mistério

Determining the role of these hitherto unknown receptors became the focus of the research, which began during Thiago Nakahara's master's degree: “The first step was to describe these neurons: who are the cells that express these genes? How many cells are there per individual? And, above all, what is the function of these cells?”, explained Papes. “This is perhaps one of the most laborious, complicated tasks in biology: finding something new in a living being and asking the question 'What is it for, what is its function?' ”

“We tested known stimuli for this vomeronasal organ, such as the smell of the opposite sex: I exposed the male to the female, the female to the male, I exposed a male to another (male), to see if this neuronal population was activated. And I also tested other things, such as phobiogenic stimuli, which cause fear, such as snake odor and cat odor”, reported Nakahara. “And none of these were able to activate this sensory population. I also tested molecules that are not related to this specific organ, but to the body's main olfactory system: several common odorants. No results".

One of the steps taken by Professor Papes' team was to determine whether these receptor cells were already present even in the animals' childhood. “With most olfactory receptors, this is true: they are expressed already during embryonic development,” Papes said. “When the animal is born, it already has a large part of its sensory repertoire ready. It makes sense: it is already prepared to respond to the world.”

“But with regard to these specific cells, something surprising happened: they are not present when the animal is born, they only appear when the animal becomes an adult,” he added. “So, it is a different population of cells, a receptor that is different from all the others known. These cells are only present in adults, not in puppies or in embryonic development.”

Weaning or aggression

The presence of a temporal pattern of gene expression may be related to its function. “It makes sense to hypothesize the role of these cells acting in some different way in adults and puppies,” said Papes. “With this in mind, we raised some hypotheses.”

“One hypothesis is that it could be related to the detection of milk, leading to the generation of some type of repugnance towards it”, explained Thiago. According to the group, this idea makes sense, because at a certain point during development the puppies stop looking for their mother and look for food on their own. Maybe because the smell of milk becomes unpleasant? “That was a possibility,” reported Papes. “Are these receptors, in adults, involved in weaning? Do they start to detect milk as a bad sign?” However, this was also not the case: the group did not find activation of new cells by the odors of milk obtained from lactating female rodents.

Finally, after testing other possibilities that distinguish puppies from adults, such as sexual behavior, the researchers hypothesized that the olfactory cells described in the study detect puppy odors. It was already known that, in most animals, the behaviors displayed by parents towards their offspring depend on odors, both the nurturing behavior displayed by mothers (and sometimes fathers), and the aggressive behavior of virgin males towards their offspring. . “The individual, once maturity is established, but when it is still a virgin, will kill the puppies it finds”, recalled Thiago. “But an individual that has already had its young does not kill the young, even if they belong to other males.” Finally, the analysis of the vomeronasal organ of virgin males exposed, with an aggressive reaction, to puppies revealed the activation of the studied cells. Active neurons were visualized in the nasal cavity by identifying a gene that is always expressed in active sensory cells, which only occurred in virgin males in the presence of puppy odors. 

Future

 “The only context in which the cells that Thiago discovered are activated is in virgin males. Fathers do not exhibit activation of these cells, nor do females, mothers or virgins. And, as he said, this is a context in which there is infanticidal, aggressive behavior. There’s a good correlation there,” Papes said. “When there is no behavior, as in fathers, mothers or virgin females, the organ is silent. The neurons are still there, they are present in females, for example. But they are not activated at all.” On the other hand, in situations where aggressive behavior is generated, the cells studied are activated. “The next step is to study why this happens,” says the professor.

“The second thing we intend to do in the future is to investigate what happens when we remove the cells we discovered,” he continued. Professor Papes' group, in collaboration with Dr. José Xavier Neto, from the National Biosciences Laboratory (LNBio-CNPEM), neighboring Unicamp, generated a transgenic mouse that does not have these receptors. “We will evaluate what behavioral change happens,” he says.

Papes recalled that the laboratory already has access to a transgenic mouse in which all sensory neurons in the vomeronasal organ are deactivated. “This mouse has a series of behavioral defects, a series of behaviors that it does not perform well: for example, gender discrimination behavior is affected. The male not only mates with females, but also with males, and the behavior of the females also changes, as they begin to actively seek out males and copulate with each other”, described Papes. The group demonstrated that virgin males of these mutant mice do not commit infanticide.

In his doctorate, Thiago studies the mechanism of modulation of smell by hormones. “In individuals who have already had litters, the sensory population that we discovered is switched off. The cells are present there, I can detect them, but they are not activatable by the puppies. Why? Is it a mechanism that has to do with the sexual experience, which releases hormones?”, explains the student. “Why are the cells that detect offspring not functional, or not playing the role of creating a behavioral response in the individuals that were fathers, or in the females? What mechanism causes these cells to shut down?”

Human

The advisor said he believes these results could have implications for the study of human sensory systems. “The vomeronasal organ was described for the first time in humans,” he said. “It is known as Jacobson's organ”, the name of the anatomist who described it, Ludwig Lewin Jacobson (1783-1843). “Very little is known about the exchange of chemical signals that exists between fathers, mothers and children,” he added.

“As in the human case the visual and auditory systems are the main sensory systems, it is assumed that most of the signals exchanged between children and their parents pass through these systems. But it is an assumption without any type of scientific proof. It is possible that the exchange of signals is much more extensive, including even odors. The human olfactory sensory system is present, as complex as in mice, and there is evidence that it affects behaviors and emotions.”

He cites as an example a classic study, published in 1995, in the journal Proceedings of the Royal Society of London, authored by Claus Wedekind, who demonstrated that women preferred the smell of t-shirts that had been worn by men with immune system characteristics different from theirs. According to Papes, this phenomenon has biological significance, as women who have children with men who are genetically different from themselves will have children with a more efficient immune system.

According to Professor Papes, other behavioral changes derived from the action of the olfactory system may be at work in humans, including in interactions between parents and children, a topic that, he believes, will represent an important field of neuroscience in the coming decades.