Microglia, Marijuana, and the Munchies

As much as we know about the human body today, it’s still mind blowing to think that microscopic cells and molecules sustain our lives. I’m here to tell you about a type of cell that isn’t discussed in a general biology class and is directly involved in forming brain sex differences: microglia.

My previous blog post unfolded the 2010 publication from Dr. Margret McCarthy’s lab, which ultimately showed that the endocannabinoid system (eCB) was regulating the number of astrocytes in the amygdala. Now, ten years later, her research group published a paper that seems to explain the mechanism by which cell number is being reduced in the amygdala. The group set out to investigate microglia as a link between developmental sex differences and the endocannabinoid system.

What are microglia? Just look at the photo above. These innate immune cells are present in the brain and have been previously linked to brain development. They commonly phagocytose, i.e., engulf and digest, other cells and protect the brain. In this photo, the cell on the left is a non-phagocytic microglial cell, while the cell on the right is phagocytic. Notice that the distinguishing factor between the two is that the phagocytic cell has little circles on it (the ones the arrows are pointing to). These are phagocytic cups and they are what perform the actual engulfing and breaking down. What really drew Dr. McCarthy’s group to microglia is the fact that they have CB1 and CB2 receptors. In the 2010 publication, the research revolved around the function of these cannabinoid receptors, part of the eCB, and how they may have contributed to sex differences in the brain.

The first experiment done was to determine the number of phagocytic microglia present in male and female rat pup amygdalae. To do this, they used an antibody to recognize a specific molecule, Iba1, present on all microglia. As exhibited in the graph above on the left, they found more microglia engaged in phagocytosis in males than females. However, around postnatal day 6 (P6), the number of phagocytic microglia becomes equal between the sexes. This meant that the sexual differentiation of the brain was taking place between P0 and P4, when the males had nearly double the amount of phagocytic microglia. They then wanted to do a base comparison of total microglia in the amygdala. As indicated in figure F above, juvenile males and females actually have the same number of microglia present in the amygdala. Figure E shows that the males simply have a higher percentage of phagocytic microglia. So if the total number of cells is the same, why do males need more phagocytic microglia? What are the phagocytic cups engulfing?

The researchers went right to the source and began by measuring the diameter of phagocytic cups in male and female microglia. They found no size difference between the two sexes, meaning that the microglia were eating objects consistent with their size. This eliminated synapses and other large debris from being the prey of microglia. To make sure it was cell bodies being engulfed, they used DAPI to label cells and then observed the phagocytic cups a day or two after. DAPI specifically stains DNA inside of cells. As seen in figure D, the majority of the phagocytic cups contained nuclear material in both males and females. From this they knew that the microglia were not engulfing older cells, so they looked to newborn ones. To look for newborn cells they immunolabeled the cells for PCNA. PCNA is a marker for cells that recently divided and underwent mitosis. Again, they analyzed the cups and found that male and female amygdala were consuming newborn cells. Figure H shows that male microglia engulfed a slightly higher percentage of newborn cells. However, the researchers noted that the cells being phagocytosed could have been destined for apoptosis, cell programmed death. To confirm that the cells in the cups were viable, they visualized cleaved caspase 3 (cCasp3+) which indicates that a cell was programmed to die. The results, in the figure above on the right, showed that cCasp3 was only found in 10% to 15% of phagocytic cups. There were significantly more cells with PCNA in the amygdala, indicating that difference in newborn cell number was not heavily influenced by apoptosis.

The next big question was if the sex differences were due to androgen receptors or estrogen receptors. To test it, they treated female pups on postnatal day 0 with a masculinizing dose of testosterone or a combination of testosterone and flutamide. Flutamide is an androgen receptor antagonist, meaning that it would turn off the androgen receptors and prevent them from processing testosterone. In order to compare the effects, they measured the resulting levels of two different cannabinoids, 2-AG and AEA. Figure B shows that testosterone in females lead to the same levels of 2-AG as seen in a normal male, while the combination of testosterone and flutamide lead to 2-AG levels equal with the control female. Figure C shows that the experiment had no effect on the AEA levels. Just as in the 2010 experiment, the group used BrdU as a marker for newborn cells and compared the number of newborn cells in both sexes. As seen in figure E, testosterone caused the female pup to have the same number of newborn cells as a control male pup. A masculinizing dose of testosterone also resulted in the female pups having more phagocytic microglia in their amygdala (Fig. F).The conclusion from these four figures was that testosterone and androgen receptors are what initiate the pathway to sexually differentiate the male brain.

It was well known prior to even the 2010 publication that there is a parallel between the eCB system and testosterone; an increase in testosterone leads to an increase in the work of the endocannabinoid system. The group hypothesized that the eCB system directly induced phagocytosis so they set out to find out how the microglia were being conducted. They performed a similar experiment to one they did for the 2010 publication, but using advancements from the past decade. In the earlier paper, the group used the WIN agonist to activate the CB1/CB2 and used a subsequent antagonist to confirm that CB2 receptors were responsible for the death of newborn cells. However, the WIN agonist was nonselective, meaning that it was said to work equally on both receptors and this experiment in the 2019 paper seems to disprove those results. Instead of using a nonselective agonist, a chemical that activates a specific target, they chose to use agonists that individually target the receptors. ACEA is a CB1 agonist and GP1a is a CB2 receptor agonist. As seen above, the females were either treated with one agonist or a combination of both. The graph on the left uses BrdU again and shows that treatment with any of the individual agonists or the combination leads to a reduced number of newborn cells in the female amygdala. The same happens in the bar chart on the right, treatment with the agonists sees an increase of phagocytic microglia to the same levels of a control male. Based on the results from the first paper, only GP1a, the CB2 agonist, should have had effects on the numbers of newborn cells and phagocytic microglia. The graph on the far right explains the results best, as the number of phagocytic microglia increases, the number of newborn cells decreases.

The last thing the group did to confirm their previous conclusions was treat neonatal females with masculinizing doses of just testosterone or testosterone combined with CB1/CB2 receptor antagonists and analyze socialization. They hypothesized, and were correct, that the testosterone treatment masculinized social play in females to the same level seen in males. As for the combination treatment, the lack of social play confirmed that it was eCB signaling that is necessary for androgen mediated masculinization of rough and tumble play. Dr. McCarthy combines the results of the the two publications at the end when she says “We identify the amygdala as an essential node mediating the sex difference in juvenile social play, which is established developmentally by the phagocytic action of microglia. Testosterone-induced endocannabinoids drive microglia phagocytosis of newborn astrocytes, which ultimately...promotes a higher frequency of play.”

One of the amazing implications that the group draws from this research concerns cannabis use by pregnant women. Since THC and other cannabinoid molecules can cross the placenta and into the fetus, there is a chance for influence in brain development. Prenatal-cannabis studies have shown subtle effects on brain development already, and the results of this paper prove how easy it is to manipulate the various pathways. Studies such as this present the chance to better understand biological pathways and the possible side effects of an increasingly legal drug.