If we test the offspring of these different mothers when the pups are older and independent, an interesting effect emerges. When we challenge these now adult rats with a mildly stressful situation, the ones that were licked and groomed the most stay fairly calm. The ones that were relatively deprived of ‘mother love’ react very strongly to even mild stress. Essentially, the rats that had been licked and groomed the most as babies were the most chilled out as adults.
The researchers carried out experiments where newborn rats were transferred from ‘good’ mothers to ‘bad’ and vice versa. These experiments showed that the final responses of the adults were completely due to the love and affection they received in the first week of life. Babies born to mothers who were lacklustre lickers and groomers grew up nicely chilled out if they were fostered by mothers who were good at this.
The low stress levels of the adult rats that had been thoroughly nurtured as babies were shown by measuring their behaviour when they were challenged by mild stimuli. They were also monitored hormonally, and the effects were as we would expect. The chilled-out rats had lower levels of corticotrophin-releasing hormone in their hypothalamus and lower levels of adrenocorticotrophin hormone in their blood. Their levels of cortisol were also low, compared with the less nurtured animals.
The key molecular factor in dampening down the stress responses in the well-nurtured rats was the expression of the cortisol receptor in the hippocampus. In these rats, the receptor was highly expressed. As a result, the cells of the hippocampus were very efficient at catching even low amounts of cortisol, and using this as the trigger to subdue the downstream hormonal pathway, through the negative feedback loop.
This showed that levels of the cortisol receptor stayed high in the hippocampus, many months after the all-important licking and grooming of the baby rats. Essentially, events that only happened for seven days immediately after birth had an effect that lasted for pretty much all of a rat’s life.
The reason the effect was so long-lasting is that the initial stimulus – being licked and groomed by the mother – set off a chain of events that led to epigenetic changes to the cortisol receptor gene. These changes occurred very early in development when the brain was at its most ‘plastic’. By plastic, we mean that this is the time when it’s easiest to modify the gene expression patterns and cellular activities. As the animals get older, these patterns stay set in place. That’s why the first week in rats is so critical.
The changes that take place are shown in Figure 12.2. When a baby rat is licked and groomed a lot, it produces serotonin, one of the feel-good chemicals in mammalian brains. This stimulates expression of epigenetic enzymes in the hippocampus, which ultimately results in decreased DNA methylation of the cortisol receptor gene. Low levels of DNA methylation are associated with high levels of gene expression. Consequently, the cortisol receptor is expressed at high levels in the hippocampus, and can keep the rats relatively relaxed[209].
This is a very interesting model to explain how early life events can influence long-term behaviour. But it seems unlikely that just one epigenetic alteration – even one as significant as DNA methylation levels at a very important gene in a critical brain region – could be the only answer. Five years after the work described above, another paper was published by a different group. This also showed the importance of epigenetic changes but in a different gene.
The later group used a mouse model of early-life stress. In this model, baby mice were taken away from their mothers for three hours a day, for the first ten days of their lives. Just like the baby rats that hadn’t been licked or groomed much, these babies developed into ‘high-stress’ adults. Cortisol levels were increased in these mice, especially in response to mild stress, just like the relatively neglected rats.
The researchers working on the mice studied the arginine vasopressin gene. Arginine vasopressin is secreted by the hypothalamus, and stimulates secretion from the pituitary. It is shown in Figure 12.1. The stressed-out mice, those that had suffered separation from their mothers in early life, had decreased DNA methylation of the arginine vasopressin gene. This resulted in increased production of arginine vasopressin, which stimulated the stress response[210].
Figure 12.2 Strong nurturing of baby rats sets up a cascade of molecular events that result in increased expression of the cortisol receptor in the brain. This increased expression makes the brain very effective at responding to cortisol and down-regulating stress responses via the negative feedback loop described in Figure 12.1.
The rat and mouse experimental studies show us two important things. The first is that when early life events lead to adult stress, there is probably more than one gene involved. Both the cortisol receptor gene and the arginine vasopressin gene can contribute to this phenotype in rodents.
Secondly, the studies also show us that a particular class of epigenetic modification is not in itself good or bad. It’s where the modification happens that matters. In the rat model, the decreased DNA methylation of the cortisol receptor gene is a ‘good’ thing. It leads to increased production of this receptor, and a general dampening down of the stress response. In the mouse model, the decreased DNA methylation of the arginine vasopressin gene is a ‘bad’ thing. It leads to increased expression of this hormone and a stimulation of the stress response.
The decreased DNA methylation of the arginine vasopressin gene in the mouse model occurred through a different route to the one used in the rat hippocampus to activate the cortisol receptor gene.
In the mouse studies, separation from the mother triggered activity of the neurons in the hypothalamus. This set off a signalling cascade that affected the MeCP2 protein. MeCP2 is the protein we met in Chapter 4, which binds to methylated DNA and helps repress gene expression. It’s also the gene which is mutated in Rett syndrome, the devastating neurological disorder. Adrian Bird has shown that the MeCP2 protein is incredibly highly expressed in neurons[211].
Normally, MeCP2 protein binds to the methylated DNA at the arginine vasopressin gene. But in the stressed baby mice, the signalling cascade mentioned in the previous paragraph adds a small chemical group called a phosphate to the MeCP2 protein and because of this MeCP2 falls off the arginine vasopressin gene. One of the important roles of MeCP2 is attracting other epigenetic proteins to where it is bound on a gene. These are proteins that all cooperate to add more and more repressive marks to that region of the genome. When the phosphorylated MeCP2 falls off the arginine vasopressin gene, it can no longer recruit these different epigenetic proteins. Because of this, the chromatin loses it repressive marks. Activating modifications get put on instead, such as high levels of histone acetylation. Ultimately, even the DNA methylation is permanently lost.
Amazingly this all happens in the mice in the first ten days after birth. After that, the neurons essentially lose their plasticity. The DNA methylation pattern that’s in place at the end of this stage becomes the stable pattern at this location. If the DNA methylation levels are low, this will normally be associated with abnormally high expression of the arginine vasopressin gene. In this way, the early life events trigger epigenetic changes which get effectively ‘stuck’. Because of this, the animal continues to be highly stressed, with abnormal hormone production, long after the initial stress has vanished. Indeed, the response continues long after the animal would even normally ‘care’ about whether or not it has its mother’s company. After all, mice are not renowned for hanging about to look after their ageing parents.