Resisting cocaine: when the tablet is not wiped clean.

TLDR: Male rats that shoot up cocaine for 60days sire male (but not female) pups that are more resistant to cocaine addiction. This trait is probably passed through chemical changes to sperm DNA. I argue why this may not be the case.

For many years now scientists have known that certain things in the environment can cause long-lasting changes in the expression of an individual’s genetic material. This type of imprinting is often referred to as “epigenetics”, or “above genetics”. What is actually is, at the molecular level, is chemical changes to either the DNA itself or the proteins (called histones) that DNA wraps itself around for tighter packaging.  Since these chemical modifications can influence which genes are expressed and to what level, in a sense they are the gatekeepers to any observable characteristics you see in an individual (called “phenotype”). Indeed, epigenetics has often been called the software to your DNA hardware, and may be involved in lots of brain processes, such as maintaining memory or making a drug addict more prone to relapse.

Whether these chemical imprints can be inherited in a Lamarckian manner is still hotly debated. There’s little argument that the quality and care of parental care can cause epigenetic (and subsequent behavioral) changes in their offspring. However, long jargon warning, transgenerational epigenetic inheritance of behavior through gametes, discussed in this paper, is a totally different animal. What that giant phrase means is this: environmental stimuli cause specific chemical modifications to both DNA/histones in the parent’s brain and sperm, and this specific change is transferred from sperm to the embryo, survives through embryonic development, and finally results in changes in the expression of offspring DNA in the brain (and/or elsewhere), and changes in behavior that we can observe.

Sound impossible? This is in fact what this paper is proposing.

Epigenetic inheritance of a cocaine resistance phenotype. Vassoler et al. Nature Neuroscience 16, 42-47 (2013)  (yeah older paper, life got in the way).

In this study, the researchers trained male rats to lever-press for cocaine injections for 60 days, which is the length of time required for sperm to generate and mature. After 60 days, the males were mated with “naïve” females (females who haven’t been exposed to drugs) and together they sired many pups (Cocsired). Males that got saline instead of cocaine were also mated, and gave birth to the Salsired rats. When the younger generation was roughly 60 days old, the researchers checked to see if these naïve rats (who’ve never experienced a cocaine high) would happily shoot up cocaine by pressing a lever.

Surprisingly, male pups whose fathers had lever-pressed for cocaine (Cocsired) consumed less cocaine in total than males whose fathers only got saline (Salsired).  They were also unwilling to work harder for the coke. The daughters from those two types of dads didn’t show any difference in cocaine preference.

So what’s going on? Perhaps Cocsired males can’t learn to press a lever or they have something wrong with their reward system? To test this, the authors trained Cocsired and Salsired males to self-administer sucrose (sugar water), and there was no difference between the groups. This means Cocsired rats can learn the task just fine. What about maternal care then? It’s known in evolutionary biology that moms may change the amount of maternal care given to her offspring based on the father’s quality. However, after spying on moms nursing, grooming, and petting their sons, the researchers didn’t find a difference there either.

So they turned to epigenetics. In a previous study, the same group of scientists also trained rats to shoot up cocaine by pressing a lever.  After removing cocaine, forcing rats to go abstinent, they discovered a type of chemical change –acetylation-to histones associated with a protein called BDNF in the prefrontal cortex, which resulted in the increased expression of BDNF, and decreased the rats’ cocaine-seeking behavior (specifically, nerd warning, increased binding of transcription factor pCREB and decreased binding of MeCP2 to exon 4 of BDNF resulted in acetylated histone 3 protein, an open chromatin conformation and increased transcription).

Since Cocsired rats seem to be more cocaine resistant, the researchers hypothesized the same epigenetic changes may be present in the Cocsired male (but not female rats). This is indeed what they found! The level of BDNF, which confers cocaine resistance in drug-experienced rats, was also raised in Cocsired male rats but not female rats. BDNF seems to play a major role here - blocking the BDNF receptor made the Cocsired male rats behave like Salsired rats, as in they tend to crave cocaine more.

So it seems like somehow, cocaine is inducing epigenetic changes in the parent that gets passed on to male offspring. What could be the medium? … Sperm, of course! The authors extracted sperm from dad rats exposed to cocaine, and found an increase in acetylation in ALL histone proteins (lots of genes are in a transcriptionally active state), as well as a specific increase in the acetylation levels of histones associated with BDNF. The latter, as you may remember, was found in Cocsired male rats as well as in the prefrontal cortex of the dad rats.

So the conclusion is this: cocaine exposure results in increase transcription of BDNF in the prefrontal cortex and sperm in paternal rats, and the chemical changes that result in this tendency is passed on to the male offspring through sperm.

In all, this is a very interesting phenomenon. The idea that a specific behavior trait – cocaine resistance – can be altered in the second generation through changes in the epigenome in the sperm of parents is a mind-blowing hypothesis. However, a lot of questions remain.

First, BDNF is not only expressed in the prefrontal cortex, it is also present in the “reward” areas of the brain, and it’s been shown that similar epigenetic changes occur there following cocaine self-administration, which also result in an increase in BDNF – but!- enhancing cocaine-seeking behavior. This is completely opposite to its effect in the prefrontal cortex. So it would be interesting to see if there are any changes in the epigenome in the reward areas of the brain of Cocsired male rats, and if so, why those changes are not influencing behavior.

Second, while the authors ruled out changes in maternal nursing behavior, it’s hard to say if the moms are extra stressed out when mating with a male rat going through cocaine-withdrawals. Stress in the mother can also result in epigenetic changes in the offspring, hence, this should be addressed, perhaps through in vitro fertilization. Indeed, in a study that showed stressed out dads can result in behavioral changes in the offspring, those changes were eliminated when i.v.f was used (Dietz DM et al).

Third, mechanistically, it is difficult to conclude these changes were the result of passage through sperm. While the same epigenetic modifications were found, this is correlational, not causational. In fact, during spermatogenesis, ~90% of all histones (where the changes in this study were found) are replaced with protamines, essentially wiping out any epigenetic marks. During development, embryos go through two rounds of “genetic reprogramming”, where most epigenetic changes are wiped out to ensure proper development of the fetus. While it is true some changes may remain, those are often associated with inhibition of DNA transcription, not activation of transcription as was found in this study.

Fourth, the lack of outliers (Cocsired male rats that behave just like Salsired male rats) is worrisome. Epigenetic changes tend to be spars by nature – a lack of outliers would mean that all the sperm that went on to generate the Cocsired male rats had the modification. However, epigenetic tagging may not occur in ALL the sperm (in fact this is rather unlikely).

Finally, how does cocaine act on the sperm to induce the SAME epigenetic change as seen in the brain? We know that cocaine can directly bind to an unidentified protein on the sperm. But it is rather amazing that this binding can eventually result in the same epigenetic tag.

Overall this is an incredibly interesting phenomenon. Hopefully the authors can follow up with more mechanism, and boundary conditions – as in, how long, how much, how frequently would the father rats have to administer cocaine to see a change? Can it be passed on further to the third (fourth) generation? Why is this effect not seen in female offsprings?

Discoveries await!