Boys will be boys

For six consecutive summers as a teenager I worked as a camp counselor. I watched over the same age group of kids each year: three to five year olds. I can vividly describe instances from each summer where I was forced to break-up some pretty violent fights. You might not believe it, but once I even had to restrain a little boy from trying to lick another boy to death! As a general rule, I noticed that juvenile boys tend to engage in rough play much more often than juvenile girls. Amazingly, during the 1970s, a couple of research groups recognized the same pattern across other mammalian species. For example, several species of primates and rats show social behavior differences between males and females. The young males of mammalian species consistently engage in more play-fighting, while the juvenile females engage in more social grooming and play-mothering. So why do juvenile males play fight more than females? A paper published several years ago by Dr. Margret McCarthy’s group at the University of Maryland gave us a clue into the molecular and cellular mechanisms that explain why juvenile male mammals like to bite, hit, kick, and punch each other more so than juvenile females.

Thanks to the research done by Dr. Michael Meaney and others in the early 1980’s, it’s been well documented that the amygdala is critical for “modulating sex differences in juvenile rough and tumble play and regulation of adult social behaviors, including mating, parenting, aggression, and territoriality.” It was previously known that sex differences in mammals were generally due to gonadal hormones, meaning a surge in testosterone from the testes around birth leads to the organization of a male brain so that males display male behaviors. At the time, the amygdala was known to be an important neuroendocrine control area in the brain. The amygdala is also a sexually dimorphic region of the brain, meaning that it is slightly different between males and females. Importantly, the amygdala had also been implicated in aggressive behaviors among adult mammals. These observations led Dr. Meaney’s hypothesize that the amygdala is a key site in the brain the contributes to play-fighting in juvenile males. So he and his group manipulated the amygdala in rats and studied its effect on the social behaviors of juvelines. The research found that lesions on the amygdala of juvenile male rats suppressed rough and tumble play, while lesions of the amygdala had no impact on juvenile female social play. Thirty years after this publication, Dr. McCarthy’s group set out to understand how the amygdala differs between the sexes and what might regulate the difference.

To begin answering these questions, McCarthy’s group began by using a nucleotide, BrdU, to mark newborn cells of the amygdala in male and female rat pups. As seen in the figure below, rats injected with the “vehicle” (i.e., the control) showed a greater number of newborn cells in juvenile females than juvenile males. This led to the question of, how is this difference in newborn cell number regulated? The research group knew from ten years of prior studies that the endocannabinoid system (eCB), is heavily involved in processes like memory, immune functions, and reproductive functions. The eCB system utilizes cannabinoid receptors which were also known to be crucial in early brain development. To see if and how the eCB regulates cell proliferation, the group treated newborn pups with WIN, an endocannabinoid receptor agonist. This chemical targets the CB1 and CB2 receptors of the eCB and activates them. Lo and behold, administering the WIN to the female pups led to a more active eCB and, as a result, a reduction in the number of newborn cells. Comparing it with the males, as seen on the right side of the figure below, the agonist had no effect on the number of newborn cells. This meant that the eCB system normally acts in males to reduce the number of cells in the amygdala!

As you can guess, these results led to another question: which receptor, CB1 or CB2, is responsible for the endocannabinoid system’s effects in males? To answer this, they did the exact same experiment described above, but simultaneously added a CB1- or CB2-specific antagonist. This chemical would deactivate its target receptor and allow for a comparison of the individual receptors. As seen in the figure below, the combination of the eCB agonist and the CB1 antagonist saw no difference compared to the experiment with just the agonist. However, the combination of the WIN and CB2 antagonist saw a significant increase in the average number of newborn cells in female amygdalae. This meant that inhibiting CB2 reversed the effects from the eCB agonist, thus concluding that the CB2 receptors were responsible for death of the newborn cells.

The last question they had with regards to the mechanism was, what type of neuronal cells in the amygdala were proliferating more in females than males? The immediate thought would be neurons. Interestingly, it was actually a type of glial cell called an astrocyte. Glial cells play a crucial part in the central nervous system by supporting and protecting neurons. Once again, McCarthy’s group performed the exact same experiment with the BrdU marker, but added markers for astrocytes, GFAP, and neurons, NeuN. They then allowed the rats to develop for fourteen days and then euthanized them to compare the presence of GFAP and NeuN. As seen in the figure below, there was a significant difference between the sexes with regards to GFAP expression.

Did this mean that the astrocytes were the key to understanding play fighting? Lastly, the group added the endocannabinoid system agonist and reduced the number of astrocytes in the females to that of males. They hypothesized that the play fighting would be masculinized in the females. It was!

These results suggested that the eCB system somehow regulates the difference in astrocyte cell number...but how? Stay tuned. A 2020 publication by Dr. McCarthy’s group found the answer, and it was a big surprise. As for the licking kid I mentioned at the beginning, I’m not sure science has an answer just yet but I think they’re working on it.